A polydopamine mediated amphiphilic molecular grafting approach was designed by combining hydrophilic and hydrophobic segments into the same moiety for creating amphiphilic surfaces with enhanced antifouling properties. Polydopamine (PDA) coating was performed on microporous polysulfone (PSF) membranes to form an active mediating layer. After that N,N-dimethyl ethylenediamine (DMEDA) was grafted on the PDA layer via Michael addition reaction and the tertiary amine functionality of DMEDA was reacted with 1-Iodo-1H,1H,2H, 2H-perfluorodecane to obtain amphiphilic molecular functionalization. All modification steps were confirmed by characterizations using X-ray photoelectron spectroscopy, scanning electron microscopy, capillary flow porometry, and surface wetting using a dynamic contact angle method. The antifouling study by protein adsorption shows that the hydrophilic-hydrophobic molecular layers in proximity effectively prevent nonspecific protein adsorption compared to the virgin PSF membrane. The antifouling property was further evaluated by real-time cyclic filtration with a 5 mg ml−1 bovine serum albumin (BSA) solution. The final modified amphiphilic surface has shown reduced irreversible protein bounded properties on and inside the membrane surfaces along with high flux recovery. Furthermore, the oil-in-water emulsion filtration shows excellent oil rejection at a high flux rate with nearly complete flux recovery. These findings indicate a promising and smart membrane surface modification technique that can be readily tailored for water filtration and separation applications.