A fluorescence observation apparatus according to an embodiment of the present technology includes a stage, an excitation section, and a spectroscopic imaging section. The stage is capable of supporting a fluorescently stained pathological specimen. The excitation section irradiates the pathological specimen on the stage with a plurality of line illuminations of different wavelengths, the plurality of line illuminations being a plurality of line illuminations situated on different axes and parallel to a certain-axis direction. The spectroscopic imaging section includes at least one imaging device capable of separately receiving pieces of fluorescence respectively excited with the plurality of line illuminations.