A fluorescent detection apparatus relates to an analysis technique for qualitatively detecting or quantifying biomolecules by producing an evanescent field on a surface of a substrate, exciting fluorescently labelled biomolecules on the substrate surface in the evanescent field, and detecting the resultant fluorescent light emitted from the biomolecules. The fluorescent detection apparatus has a configuration in which a well is provided in a surface opposing to a sample substrate of a prism, the well is filled with a matching liquid, and the matching liquid is filled between the sample substrate and the prism, thereby improving operability and providing a stable evanescent field.