The invention discloses a spectral unmixing detection method for in-vivo fluorescence. The spectral unmixing detection method comprises the following steps: acquiring a to-be-analyzed in-vivo fluorescence image, analyzing a mixed fluorescence spectrum of the in-vivo fluorescence, and establishing a linear spectrum mixing model of a current fluorescence image according to a spontaneous fluorescence spectrum and a target fluorescence spectrum; and establishing an orthogonal subspace projection of the linear spectrum mixing model, and combining with a least squares algorithm to eliminate the spontaneous fluorescence spectrum in the linear spectrum mixing model, so as to obtain an interested spectrum, namely the target fluorescence spectrum in a nonseparable manner.