A total internal reflection sample illuminating device, which is configured to introduce laser light into a slide glass such that the laser light undergoes multiple total internal reflections in the slide glass to generate an evanescent wave at a top surface of the slide glass, and to illuminate a sample, which is positioned on the top surface of the slide glass, with the evanescent wave, the total internal reflection sample illuminating device comprising: a slide glass; a laser light source; a device substrate, which has an opening formed to pierce between a top surface and a bottom surface of the device substrate, and has the slide glass placed on the top surface so as to block the opening, the device substrate being formed of a material having a refractive index at the same level as a refractive index of the slide glass such that the total internal reflection of the laser light in the slide glass occurs only at the front surface of the slide glass on which the sample is placed, and a back surface of the slide glass facing the opening; a light source holder to hold the laser light source, and which is configured to allow laser light emitted by the laser light source to enter the top surface of the device substrate at a predetermined angle; an incident reflecting surface, which is formed on the bottom surface side of the device substrate, and which is configured to reflect the laser light that has entered the device substrate from the laser light source such that the laser light enters the slide glass and undergoes multiple total internal reflections in an area of the slide glass that overlaps the opening; and an exit reflecting surface, which is formed on the bottom surface side of the device substrate, and in an area opposed to the incident reflecting surface across the opening; the total internal reflection sample illumination device being characterized in that the light source holder is fixed to the top surface of the device substrate, and in that the exit reflecting surface is configured to reflect the laser light that has entered the device substrate from the slide glass in a direction that is parallel to the top surface of the device substrate and that does not intersect with the opening. a slide glass; a laser light source; a device substrate, which has an opening formed to pierce between a top surface and a bottom surface of the device substrate, and has the slide glass placed on the top surface so as to block the opening, the device substrate being formed of a material having a refractive index at the same level as a refractive index of the slide glass such that the total internal reflection of the laser light in the slide glass occurs only at the front surface of the slide glass on which the sample is placed, and a back surface of the slide glass facing the opening; a light source holder to hold the laser light source, and which is configured to allow laser light emitted by the laser light source to enter the top surface of the device substrate at a predetermined angle; an incident reflecting surface, which is formed on the bottom surface side of the device substrate, and which is configured to reflect the laser light that has entered the device substrate from the laser light source such that the laser light enters the slide glass and undergoes multiple total internal reflections in an area of the slide glass that overlaps the opening; and an exit reflecting surface, which is formed on the bottom surface side of the device substrate, and in an area opposed to the incident reflecting surface across the opening; the total internal reflection sample illumination device being characterized in that the light source holder is fixed to the top surface of the device substrate, and in that the exit reflecting surface is configured to reflect the laser light that has entered the device substrate from the slide glass in a direction that is parallel to the top surface of the device substrate and that does not intersect with the opening.