A sample observation device includes a sample container that holds the sample stained with a first fluorescent substance and a solution including a second fluorescent substance, an irradiation unit that performs irradiation with first excitation light and second excitation light, a scanning unit that scans the sample container in one direction, an image formation unit that forms first fluorescent light from the sample and second fluorescent light from the solution, an imaging unit that outputs first image data based on the first fluorescent light and first image data based on the second fluorescent light, an image processing unit that generates a first fluorescent light image based on a plurality of pieces of first image data and a second fluorescent light image based on a plurality of pieces of second image data, and an analysis unit that specifies an area in which there is the sample on the basis of the second fluorescent light image and sets an analysis area in the first fluorescent light image.