A quantitative determination method of a biological substance in a sample stained with a staining reagent comprising fluorescent particles each encapsulating a fluorescent substance and binding to a biological substance recognizing site. The method comprises inputting a fluorescent image obtained by photographing the sample, extracting a predetermined region from the fluorescent image to calculate an integrated luminance of the predetermined region, and counting the number of fluorescent particles contained in the predetermined region from the integrated luminance and the average luminance per fluorescent particle. The average luminance per fluorescent particle is calculated from a correlation between the number of fluorescent particles counted from an image of the fluorescent particles visualized and the luminance derived from fluorescent light from the fluorescent particles and calculated from a fluorescent image of a region identical to the region taken in the image from which the number of fluorescent particles is counted.