A method and apparatus for separating and extracting main sound sources from a mixed musical sound signal are provided. A musical sound source separation apparatus may include an prior information signal compressor to compress an prior information signal including a characteristic of a predetermined sound source, a mixed signal divider to divide a mixed signal including a plurality of sound sources into a plurality of segments, a Nonnegative Matrix Partial Co-Factorization (NMPCF) analyzer to acquire common information shared by the plurality of segments, by applying an NMPCF algorithm to the prior information signal, and a target musical instrument signal separator to separate a target musical instrument signal corresponding to the predetermined sound source from the mixed signal, based on the common information.