A signal separation device for acquiring a source signal from a mixed signal observed by a plurality of sensors includes: a database that stores feature information of a clean signal; separation matrix calculation means for repeatedly performing processes of, based on a separated signal obtained by multiplication of a mixed signal converted into a time-frequency representation by a separation matrix and on the feature information stored in the database, calculating a parameter to be used for an objective function for optimizing the separation matrix, and calculating a separation matrix for minimizing the objective function using the parameter; and output means for outputting a separated signal calculated using the optimized separation matrix obtained by the separation matrix calculation means.