An illumination system for a STED optical microscope, the illumination system comprising an illumination light source, and an illumination light path composed of optical elements; wherein the illumination light source is configured to emit a light beam which, after passing through the illumination light path, is focused and irradiated onto a sample to excite fluorescent substance in the sample to emit fluorescence; wherein the illumination light path comprises a first optical filter, a second optical filter, a polarization beam splitter, a first quarter-wave plate, a first dichroic element, an optical path delay unit, a phase plate, a second dichroic element and a second quarter-wave plate, which are arranged in sequence in a transmission direction of the light path; wherein the first optical filter and the second optical filter are configured to optically filter the light beam emitted from the illumination light source, so as to obtain a first light beam and a second light beam each having a certain wavelength; wherein the polarization beam splitter is configured to respectively split the first light beam and the second light beam to form linearly polarized lights which, after being reflected, are incident onto the first quarter-wave plate and the first dichroic element in sequence; wherein the first light beam is configured to form circularly polarized light after passing through the first quarter-wave plate, and after being reflected by the first dichroic element, to passe through the first quarter-wave plate again to form linearly polarized light, which is then transmitted through the polarization beam splitter, reflected by the second dichroic element, and converted by the second quarter-wave plate into circularly polarized light to be incident onto a microscopic objective lens of a microscopic imaging system and converged, thus forming a first light spot at a focal plane of the microscopic objective lens; wherein the second light beam is configured to form circularly polarized light after passing through the first quarter-wave plate, and after being transmitted through the first dichroic element, to be incident onto the optical path delay unit and the phase plate; after being reflected by the phase plate, the second light beam is configured to be sequentially exited from the optical path delay unit, transmitted through the first dichroic element, converted by the first quarter-wave plate into linearly polarized light, transmitted through the polarization beam splitter, reflected by the second dichroic element, converted by the second quarter-wave plate into circularly polarized light, and incident onto the microscopic objective lens of the microscopic imaging system and converged, thus forming a second light spot at the focal plane of the microscopic objective lens, wherein a center of the first light spot coincides with a center of the second light spot. wherein the illumination light path comprises a first optical filter, a second optical filter, a polarization beam splitter, a first quarter-wave plate, a first dichroic element, an optical path delay unit, a phase plate, a second dichroic element and a second quarter-wave plate, which are arranged in sequence in a transmission direction of the light path; wherein the first optical filter and the second optical filter are configured to optically filter the light beam emitted from the illumination light source, so as to obtain a first light beam and a second light beam each having a certain wavelength; wherein the polarization beam splitter is configured to respectively split the first light beam and the second light beam to form linearly polarized lights which, after being reflected, are incident onto the first quarter-wave plate and the first dichroic element in sequence; wherein the first light beam is configured to form circularly polarized light after passing through the first quarter-wave plate, and after being reflected by the first dichroic element, to passe through the first quarter-wave plate again to form linearly polarized light, which is then transmitted through the polarization beam splitter, reflected by the second dichroic element, and converted by the second quarter-wave plate into circularly polarized light to be incident onto a microscopic objective lens of a microscopic imaging system and converged, thus forming a first light spot at a focal plane of the microscopic objective lens; wherein the second light beam is configured to form circularly polarized light after passing through the first quarter-wave plate, and after being transmitted through the first dichroic element, to be incident onto the optical path delay unit and the phase plate; after being reflected by the phase plate, the second light beam is configured to be sequentially exited from the optical path delay unit, transmitted through the first dichroic element, converted by the first quarter-wave plate into linearly polarized light, transmitted through the polarization beam splitter, reflected by the second dichroic element, converted by the second quarter-wave plate into circularly polarized light, and incident onto the microscopic objective lens of the microscopic imaging system and converged, thus forming a second light spot at the focal plane of the microscopic objective lens, wherein a center of the first light spot coincides with a center of the second light spot.