In acquisition of an image of cells, a focal position is accurately set at highly active cells rather than focusing on dead cells. Provided is an automatic focusing apparatus (8) used in a microscope (1) that image captures fluorescence emitted from cells to acquire a cell image, the automatic focusing apparatus (8) including a setting unit (5) that sets a luminance range indicating a region where viable cells exist on the basis of a luminance distribution of the acquired cell image; and a focus-detecting unit that detects a focal position on the basis of a luminance of an image of nuclei of the cells within the luminance range set by the setting unit (5).