The aqueous dispersion of graphene oxide (GO) was employed as additive to fabricate antifouling and antibacterial polyethersulfone (PES)/sulfonated polysulfone (SPSf)/GO mixed matrix membranes (MMMs) by the non-solvent induced phase separation (NIPS). The effect of different amounts of GO on the morphology and performance of MMMs were studied. The results showed that the casting solution exhibited an increasing trend in viscosity with increment in GO concentration (from 0 to 0.016 wt%) owing to the hydrogen bonding (H-bonding) interaction among GO, HO and SPSf. Raman and molecular dynamic (MD) simulations analyses confirmed that there existed H-bonding interaction among SPSf, GO and HO. Specifically, the agglomeration of GO was inhibited and stable homogeneous casting solution was obtained. Meanwhile, the H-bonding interaction also played a key role in the MMMs structure and improved properties. It was found that GO nanosheets were uniformly embedded to form many cellular-like voids in the asymmetric PES/SPSf/GO MMMs with a sponge-like structure. The pure water flux of the MMMs with a very low GO content of 0.012 wt% was up to 816.9 L/mh and the rejection of bovine serum albumin (BSA) was more than 99.2% under a pressure of 0.1 MPa. Additionally, the mechanical properties of MMMs was also improved with the increase of GO content. Importantly, the MMMs displayed excellent antifouling and antibacterial performance. A high fouling recovery (94.2%) and antibacterial rate (90.0%) against Escherichia coli (E. coli) obtained were attributed to improved hydrophilicity, enhanced negative charge and GO nano-size effect. In summary, our study provides a simple approach to tailor MMMs with the enhancement of permeation, antifouling and antibacterial properties at a very low content of GO.