A biological substance quantitation method which quantitates an amount of a specific biological substance in a sample stained with a staining reagent which stains the biological substance with a fluorescent substance, the method comprising: inputting a fluorescent image which represents a presence of the biological substance in the sample by a fluorescent bright spot; performing fluorescence quantitation which includes calculation of an evaluation value by quantitative evaluation of the fluorescent bright spot in the fluorescent image; inputting a standard fluorescent image under a same condition as the inputting of the fluorescent image, wherein the standard fluorescent image represents a presence of the biological substance in a standard sample by a fluorescent bright spot based on staining under a same condition as the sample, and an amount of the biological substance in the standard sample is measured in advance; performing standard fluorescence quantitation which includes calculation of an evaluation value by quantitative evaluation of the fluorescent bright spot in the standard fluorescent image under a same condition as the quantitation of fluorescence; calculating a correlation between the amount of the biological substance in the standard sample and the evaluation value of the fluorescent bright spot in the standard fluorescent image; and converting the evaluation value of the fluorescent bright spot in the fluorescent image to an amount of the biological substance in the sample based on the calculating of the correlation, characterized in that the fluorescent substance comprised by the staining reagent is a fluorescent substance-encapsulating nanoparticle in which a plurality of molecules of the fluorescent substance are accumulated, wherein the fluorescent substance-encapsulating nanoparticle has an average particle size of from 40 to 280 nm. inputting a fluorescent image which represents a presence of the biological substance in the sample by a fluorescent bright spot; performing fluorescence quantitation which includes calculation of an evaluation value by quantitative evaluation of the fluorescent bright spot in the fluorescent image; inputting a standard fluorescent image under a same condition as the inputting of the fluorescent image, wherein the standard fluorescent image represents a presence of the biological substance in a standard sample by a fluorescent bright spot based on staining under a same condition as the sample, and an amount of the biological substance in the standard sample is measured in advance; the standard fluorescent image represents a presence of the biological substance in a standard sample by a fluorescent bright spot based on staining under a same condition as the sample, and an amount of the biological substance in the standard sample is measured in advance; performing standard fluorescence quantitation which includes calculation of an evaluation value by quantitative evaluation of the fluorescent bright spot in the standard fluorescent image under a same condition as the quantitation of fluorescence; calculating a correlation between the amount of the biological substance in the standard sample and the evaluation value of the fluorescent bright spot in the standard fluorescent image; and converting the evaluation value of the fluorescent bright spot in the fluorescent image to an amount of the biological substance in the sample based on the calculating of the correlation, characterized in that the fluorescent substance comprised by the staining reagent is a fluorescent substance-encapsulating nanoparticle in which a plurality of molecules of the fluorescent substance are accumulated, wherein the fluorescent substance-encapsulating nanoparticle has an average particle size of from 40 to 280 nm.