Ultrafiltration technology has found widespread application in bioprocessing, particularly in protein concentration and buffer exchange for downstream processes. The demand for this technology has spurred research efforts to develop excellent membrane structures with superior separation functionality. However, challenges persist in transitioning laboratory research to industrial-scale continuous production processes while simultaneously optimizing membrane structure and performance through a single fabrication method. Thus, there is an urgent need for pilot-scale membrane preparation to improve membrane morphology and separation efficiency while ensuring scalability. This study investigates a continuous membrane fabrication method, systematically exploring the influence of different additives and vapor-assisted nonsolvent induced phase separation (VNIPS) on the structure and performance of polyethersulfone (PES) membranes. Evaluations of PES membranes with distinct structures were performed, employing flux decline, resistance analysis, and alkali resistance tests to distinguish their performance and bioseparation capabilities. The results showed that good sponge-like pore structure was obtained by VNIPS process, which realized the synergistic optimization of permeation and retention properties. Besides, the sponge-like porous structure membranes prepared by amphiphilic copolymer additives are more suitable for bioseparation.