There is provided a biological substance quantitation method of quantitating a biological substance in a sample stained with a staining reagent including a fluorescent particle encapsulating a fluorescent substance, based on a fluorescence of the fluorescent substance. The method includes inputting a fluorescent image representing expression of the biological substance in the sample by a fluorescent bright spot; and quantitating an expression amount of the biological substance based on a fluorescence of the fluorescent bright spot. The biological substance is a nucleoprotein expressed at a cell nucleus. The fluorescent particle binds to the biological substance through a primary antibody which is directed against the biological substance as an antigen.