Provided are an apparatus and method of separating, from a mixed signal, a sound source generated using a rhythm musical instrument based on characteristics of the rhythm musical instrument repeated in an aspect of time. The apparatus may include a separation unit to separate a plurality of mixed signals into a plurality of segments, a Nonnegative Matrix Partial Co-Factorization (NMPCF) analysis unit to perform an NMPCF analysis on the plurality of segments, and to obtain a plurality of entity matrices based on the analysis result, a target instrument signal separating unit to separate, from the mixed signals, a target instrument signal, by calculating an inner product between the plurality of entity matrices, and a signal association unit to associate the target instrument signals separated from each of the plurality of segments.