The separation efficiency of currently developed bacteria-removal membrane system will be limited and lost due to the chemical cleaning and regeneration procedure. The main innovation of this research is the development of a novel intelligent bacterial filtration process to produce a polycationic membrane separation system that continuously kills and releases bacteria even after a 121 °C sterilization procedure. This work breaks through the possibility of introducing medical-grade sterilization procedures for clinical applications while providing the implementation of self-cleaning techniques that can be facilitated by sustainable cycle operation. The polycationic membrane was prepared by the vapor-induced phase separation (VIPS) process to control the membrane formation of polyvinylidene difluoride (PVDF) blended with a new copolymer of poly(butyl methacrylate-co-(trimethylamino)propyl methacrylamide chloride) (PVDF/PBTP). The tested hypothesis is to validate the effect of the chemical-segment substitution of methacrylamide for methacrylate on the bacteria-killing/releasing functions. The reversible molecular interaction between the membrane and microorganisms is driven by controlling the electrostatic attraction adjusted by a multivalent negatively charged ions. The PVDF/PBTP membrane can sustainably provide more than 80% bacteria-killing efficiency after the high-pressure steam sterilization process. The results of multiple bacterial separation cycles show that the PVDF/PBTP membrane can exhibit a bacteria-killing level of more than 82% and an average bacteria-releasing level of 75% without aging under 5 repeated membrane filtrations. The significant results show that PVDF/PBTP membrane exhibit excellent bacteria-killing/releasing function proved through multiple cycles of bacterial isolation. The new self-cleaning membrane developed in this study can provide a potential strategy to effectively remove potentially harmful microorganisms in domestic water and industrial wastewater, and significantly reduce its environmental pollution and bacterial pathogenicity.