The invention relates to a mixing matrix estimation method for underdetermined blind source separation, which performs short-time Fourier transform on two received observation signals to obtain the short-time Fourier coefficients X1(t,f) of the two observation signals ) and X2(t,f) to form multiple scattered points and remove low-energy scattered points; calculate the ratio of the remaining scattered points, classify the scattered points according to the ratio, and obtain the M category with the largest number of scattered points, and calculate each Then convert the mean value into the slope angle of the straight line, and then convert it into a column vector, and obtain the rotation matrix T according to the column vectors with close directions, and perform rotation transformation to obtain a new two-way observation signal X′1( t,f) and X′2(t,f); for the two observation signals X′1(t,f) and X′2(t,f), classify the scattered points again according to the ratio of the two observation signals , get the S class with the largest number of scattered points, calculate the mean value of the ratio of scattered points in each class, and then get the similar slope angles, get the corresponding column vectors through these slope angles, and finally get the entire mixing matrix.