A total internal reflection fluorescence imaging system, comprising: a first imaging system, wherein the first imaging system comprises N separate laser light paths, an objective lens, a first receiving device, N first light sources, a reflecting mirror, N is an integer greater than one, each laser light path of the N separate laser light paths is configured to generate light to enter the objective lens, the light passing through the objective lens illuminates a sample to excite the sample to emit fluorescence, the fluorescence passes through the objective lens and enters the first receiving device to form a first image; and a second imaging system, wherein the second imaging system is an automatic focusing system, the second imaging system comprises a light emitting source, the objective lens, and a second receiving device, the light emitting source is configured to emit light to enter the objective lens, the light passing through the objective lens illuminates the sample and is reflected, the reflected light passes through the objective lens and enters the second receiving device to form a second image; characterized in that the first imaging system further comprises N first dichroic mirrors, wherein each of the N first light sources corresponds to one of the N first dichroic mirrors, wherein the N separate laser light paths are composed by the N first light sources, the reflecting mirrors, and the N first dichroic mirrors, and wherein light generated in each of the laser light path is emitted from the first light source, the light emitted from the first light source is reflected by the reflecting mirror to enter the first dichroic mirror, the first dichroic mirror reflects the light into the objective lens. a first imaging system, wherein the first imaging system comprises N separate laser light paths, an objective lens, a first receiving device, N first light sources, a reflecting mirror, N is an integer greater than one, each laser light path of the N separate laser light paths is configured to generate light to enter the objective lens, the light passing through the objective lens illuminates a sample to excite the sample to emit fluorescence, the fluorescence passes through the objective lens and enters the first receiving device to form a first image; and a second imaging system, wherein the second imaging system is an automatic focusing system, the second imaging system comprises a light emitting source, the objective lens, and a second receiving device, the light emitting source is configured to emit light to enter the objective lens, the light passing through the objective lens illuminates the sample and is reflected, the reflected light passes through the objective lens and enters the second receiving device to form a second image; characterized in that the first imaging system further comprises N first dichroic mirrors, wherein each of the N first light sources corresponds to one of the N first dichroic mirrors, wherein the N separate laser light paths are composed by the N first light sources, the reflecting mirrors, and the N first dichroic mirrors, and wherein light generated in each of the laser light path is emitted from the first light source, the light emitted from the first light source is reflected by the reflecting mirror to enter the first dichroic mirror, the first dichroic mirror reflects the light into the objective lens.