PROBLEM TO BE SOLVED: To provide a fluorescence observation method, a fluorescence observation device, and a light shading member capable of conveniently executing a quantitative estimation of a fluorescence image and capable of accurately identifying an observation object.SOLUTION: This fluorescence observation method includes a fluorescence imaging step of imaging the fluorescence from an observation object P by an imaging unit 10. The fluorescence imaging includes: a step of making a dark room space between the observation object P and an observation window R by abutting a top end part 51b of a light shading member 50 against the observation object P or a mounting surface 100 of the observation object P in a state that a base end part 51a of the light shading member 50 having a cylindrical main body 51 configured to surround the observation window R of the imaging unit 10 is mounted on the imaging unit 10； and a focus adjustment step of adjusting a focus position of an imaging camera 13.SELECTED DRAWING: Figure 4【課題】蛍光像の定量的な評価を簡便に実施でき、かつ観察対象を精度良く識別できる蛍光観察方法、蛍光観察装置、及び遮光部材を提供する。【解決手段】この蛍光観察方法は、観察対象Ｐからの蛍光を撮像ユニット１０によって撮像する蛍光撮像ステップを備える。蛍光撮像ステップは、撮像ユニット１０の観察窓Ｒを囲うように筒状の本体部５１を有する遮光部材５０の基端部５１ａを撮像ユニット１０に取り付けた状態で、観察対象Ｐ又は観察対象Ｐの載置面１００に遮光部材５０の先端部５１ｂを突き当てることにより、観察対象Ｐと観察窓Ｒとの間の空間を暗室にする暗室化ステップと、撮像カメラ１３のフォーカス位置を調整するフォーカス調整ステップと、を含む。【選択図】図４
