A quantum yield calculation method uses a quantum yield calculation program for a spectrophotofluorometer. When a quantum yield is calculated using a spectrophotofluorometer 1, a calibration processing unit executes the processing to calibrate a photon number A2 that is a photon number of the fluorescence in a blank measurement state based on a photon number A1 that is the photon number of an excitation light in the blank measurement state and a photon number B1 that is the photon number of an excitation light in the sample measurement state. A quantum yield calculation processing unit calculates a first quantum yield based on a background photon number A2′ after a calibration in addition to the photon number A1 of the excitation light in the blank measurement state and the photon number B2 of the fluorescence in the sample measurement state.