A technique and device for fluorescence observation with good operability, high sensitivity, acid high reliability. The device is used for fluorescence observation using evanescent light. The angle of incidence of the excitation light is adjusted so that the excitation light is always totally reflected from the surface of a substrate irrespective of the angle of the surface of the substrate. The method includes a step of shining the excitation light on the observation substrate while continuously varying the angle of the excitation light with respect to the observation substrate, a step of sensing the shone excitation light by means of optical sensors, and a step of setting the angle of total reflection according to the result of the sensing by the optical sensors. The direction in which the shone excitation light travels varies with the angle of incidence. That is, the excitation light travels as the transmitted light, the reflected light, or the surface propagating light. These lights are sensed by the corresponding optical sensors, and how the angle of incidence of the excitation light is with respect to the critical angle is determined. The angle of incidence of the excitation light is varied depending on the result of the determination, thereby realizing an optimum total reflection angle.