Provided are a concentration measuring method of an optically active substance and a concentration measuring device of an optically active substance, which can easily and accurately measure a concentration of the optically active substance in aqueous humor. The concentration measuring method of an optically active substance includes: a first step of measuring a polarization state of a first reflected light that is obtained by irradiating an aqueous humor in an eye with an incidence light which is polarized and reflecting the incidence light at an interface between the aqueous humor and a lens, in which the polarization state of the first reflected light is measured by irradiating a first incidence light such that an angle between a normal line to a point where the incidence light intersects a surface of the lens, and the incidence light is equal to or smaller than a Brewster angle; a second step of measuring a polarization state of a second reflected light by irradiating with a second incidence light such that an angle of the incidence light is equal to or larger than the Brewster angle; a third step of calculating an optical rotation of the aqueous humor with information on the polarization state of the first reflected light and information on the polarization state of the second reflected light; and a fourth step of calculating a concentration of an optically active substance in the aqueous humor from the optical rotation of the aqueous humor.