It is challenging to improve the hemocompatibility of commercial dialyzers packaging more than 10,000 fibers. Herein, we report the preparation and evaluation of a self-anticlotting Polysulfone dialyzer via an interface crosslinking approach. By modulating the conformation of sulfate, carboxyl groups and pyrrolidone (VP) in heparin-like polymers, the hemocompatibility of the dialyzer was significantly improved. It was found that the sequential structure of functional groups influenced the anticoagulant performances substantially. The hollow fiber modified by the blended polymer (Poly (N-vinyl-2-pyrrolidone-co-triethoxyvinylsilane) and poly (Sodium 4-vinylbenzenssulfonate-co-triethoxyvinylsilane-co-Acrylic acid) (P(VP-VTES)-b-P(SSNa-VTES-AA))) exhibited better hemocompatibility than the contrast membrane modified by poly (Acrylic acid-co-triethoxyvinylsilane-co -N-vinyl-2-pyrrolidone-co--co-Sodium 4-vinylbenzenssulfonate) (P(AA-VTES-VP-SSNa)). The chemical composition was determined by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectra (FTIR). The morphology of the hollow fiber membranes was observed by a scanning electron microscopy (SEM). The thrombin time (TT), plasma fibrinogen (FIB), activated partial thromboplastin time (APTT), prothrombin time (PT) and protein adsorption were measured to examine the hemocompatibility. The modified hollow fiber showed self-anticlotting properties with excellent protein resistance, remarkably prolonged coagulation time (APTT> 600 s), suppressed complement activation and nontoxicity to osteoblasts. The modified dialyzer showed practicable ultrafiltration coefficient and clearance performance, indicating its great potential application in dialysis treatment.