In this study, a novel, simple, and eco-friendly enzymatic-reaction-based approach to produce reduced graphene oxide/polyaniline (rGO/PANI) composite material was proposed. Glucose oxidase (GOx) was used as an effective catalyst producing hydrogen peroxide, in the presence of glucose, for the oxidative polymerization of aniline under ambient conditions. The prepared rGO/PANI composite was dispersed in polysulfone (PSf), and the mixed membranes were prepared by the phase inversion polymerization method. The morphology of membranes was investigated using scanning electron microscopy (SEM), atomic force microscopy (AFM), and contact angle (CA) techniques. The performance of membranes was studied in terms of salt rejection and pure water flux. The incorporation of rGO into the membrane matrix led to hydrophobic membrane surface with the enhanced macro-voids. On the contrary, the contact angle data revealed that the rGO/PANI-incorporated membrane surface is partly hydrophilic due to the PANI fibers in membrane, whereas SEM images showed the enhanced macro-voids. Membranes exhibited an improved salt rejection after rGO/PANI doping. The rGO/PANI-modified membrane loading exhibited a maximum of 82% NaCl rejection at an applied pressure of 10 bar. In addition, the results showed that the PSf-rGO/PANI composite membrane had the highest mean porosity and water flux.