Biofouling and organic fouling are intractable problems that degrade the separation performance of reverse osmosis (RO) membranes. In the present work, we report a novel guanidine-based polymer modified RO membrane with excellent antifouling and antibacterial properties. The guanidine-based polymer, namely poly (guanidine-hexamethylenediamine-PEI) (poly(GHPEI)), was first synthesized by a polycondensation method. It contained plenty of guanidine and amine groups and its minimum bactericidal concentration was measured as 12.5 mg/L. Then the poly(GHPEI) modified membrane was fabricated via polydopamine (PDA) immobilization. A series of characterizations including surface morphology, hydrophilicity and charge were performed to analyze the structure changes resulting from poly(GHPEI) modification. Permeate flux and antifouling properties were also measured to investigate the separation properties of the modified membranes. Besides, membrane antibacterial activity and antibiofouling property were evaluated through the sterilization tests against Escherichia coil and Bacillus subtilis, and the flux decline ratio after immersing the membrane into bacterial suspension, respectively. The results indicated that the grafting modification of poly(GHPEI) endowed the RO membrane with excellent antifouling and antibacterial properties.