Ethylene vinyl alcohol copolymer (EVAL) membrane has great potential for applications in protein separation and purification, but the uncontrollable distribution of grafting location when membranes are modified by the grafting method limits the membrane performance. Herein, an effective strategy for controlling the distribution of grafting location was designed to fabricate a high-performance EVAL membrane via photografting. The UV intensity through the membranes was weakened when the local concentration of the photoinitiator benzophenone (BP) on the topside of the membrane increased; thus, the grafting location inside the EVAL membrane changed from homogenous to asymmetric distribution based on the UV absorbability of BP. The grafting inside the membrane pores can be promoted when the loose and porous surface of the EVAL membrane was used as the UV-facing side. More importantly, the varied distribution of grafting location played different roles on improving the membrane performance. For protein binding, the limited convection in the membrane bed was avoided, and the desorption efficiency could be improved when the grafting location enriched inside the membrane pores. For protein filtration, the antifouling properties of the EVAL membrane were enhanced when the grafting location enriched on the topside. This research offers a novel approach to achieve controllable grafting location distribution of membranes and provides a perspective to design the high-performance EVAL membranes for protein separation.