The present disclosure discloses a dual-channel optical three-dimensional interference method based on underdetermined blind source separation, which blindly separates out, through interference data collected by a CCD camera, interference signals between surfaces of a slide under test, to solve interference signal parameters, including an interference signal amplitude-frequency and an interference signal phase-frequency. Based on a dual-channel optical three-dimensional Michelson-type interference experiment, estimation of a mixed matrix is obtained by a K-means clustering algorithm, and recovery of a source signal is achieved by a L1 norm shortest path method. It is finally achieved that laser wavenumber scanning can accurately and blindly separate out the interference signals of the four surfaces based on light intensity values collected by the CCD camera, to achieve the blind separation of the interference signals of the four surfaces.