A fluorescence sensor is constituted by: a light source, for emitting excitation light of a predetermined wavelength; a dielectric block, formed of a material that transmits the excitation light; a metal film, formed on a surface of the dielectric block; a non flexible film of a hydrophobic material, formed on the metal film at a film thickness within a range of 10 to 100 nm; a sample holding portion, for holding a sample such that the sample contacts the non flexible film; an incident optical system, for causing the excitation light to enter the interface between the dielectric block and the metal film through the dielectric block such that conditions for total internal reflection are satisfied; and fluorescence detecting means, for detecting fluorescence emitted by a substance within the sample, which is excited by evanescent waves that leak from the interface when the excitation light enters the interface.