The invention discloses a method for preparing an antimicrobial polymer separation membrane, which comprises the following steps of: (1) selecting a polymer separation membrane containing a dimethylaminoethyl methacrylate surface modification layer as a basement membrane; and (2) soaking the basement membrane in aqueous solution of silver nitrate and aqueous solution of sodium borohydride in turn, taking out, soaking in cross-linking agent solution for quaternization and in-situ cross-linking reaction, taking out, cleaning, and drying to obtain the antimicrobial polymer separation membrane, wherein a reducing agent is sodium borohydride or sodium ascorbate; and a cross-linking agent is on ore more of alpha,alpha'-dichloro-p-xylen, 1,2-dibromoethane, 1,3-dibromopropane, and 1,4-dibromobutane. The polymer separation membrane prepared by the method fully utilizes the antibacterial performance of silver nanoparticles and quaternized dimethylaminoethyl methacrylate, and has excellent antimicrobial effect.