Thin film composite (TFC) polyamide reverse osmosis (RO) membranes are naturally prone to fouling, especially the biofouling, due to their inherent physicochemical surface properties. In the current study, highly efficient anti-biofouling membrane was fabricated by assembly of poly acrylic acid and potent anti-microbial agent tobramycin on a commercial TFC RO membrane surface using the layer-by-layer technique. The successful modification was verified by XPS, and the membrane surface hydrophilicity was substantially improved as detected by contact angle measurement. Membrane prepared under optimized condition shows 18% increased water flux and slightly enhanced (0.4%) salt rejection properties. The antifouling performance is significantly improved, as the modified membrane demonstrates 37% and 26% higher flux than the virgin membrane after three-cycling fouling of bovine serum albumin and sodium alginate solution, respectively. In addition, modified membrane possesses dramatically efficient anti-microbial property, which can achieve more than 99.6% killing ratio for both the Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The modified membrane well maintains its high permselectivity after 7-d severe biofouling test. Moreover, the membrane shows excellent performance stability.