A method for generating a three-dimensional luminescence image includes setting a focal interval between two-dimensional images in accordance with localization of luminescence in a three-dimensional sample. The three-dimensional sample contains a plurality of cells prepared to be luminescent and has a three-dimensional shape. The two-dimensional images have mutually different focal planes and are acquired at the focal interval. The method further includes acquiring a two-dimensional image set including two-dimensional images at the focal interval that is set by imaging the three-dimensional sample under an unirradiated condition; and generating a three-dimensional luminescence image by combining the two-dimensional images included in the two-dimensional image set together.