The reusability of molecularly imprinted polymer (MIP)-based biosensors is severely limited by inefficient template removal suppressing in situ regeneration and surface fouling in complex matrices. While recent work demonstrated the feasibility of in situ electrostatic regeneration for MIPs, no study has yet integrated antifouling capability with regeneration ability in a single MIP platform. Here, we reported the first integration of antifouling and ultrahigh in situ regeneration capabilities in a single MIP biosensor for electrochemical cortisol detection in complex biological sweat and real pharmaceutical formulations. The interface exhibited robust antifouling capability against high concentrations of oppositely charged proteins and complex serum, which was primarily attributed to high hydrophilicity of oligochitosan-based surface. Meanwhile the highly hydrated surface effectively preserved structural integrity of biosensing surface and recognition cavities during repeated electrochemical template extraction. As a result, the biosensor achieved 23 regeneration cycles in buffer, a 4.6-fold improvement over previous report, and, more importantly, 12 cycles in sweat and over 15 cycles in pharmaceuticals. Unlike prior works that addressed only regeneration in buffer without antifouling consideration, this new platform simultaneously tackled both long-standing challenges within a single MIP architecture. The biosensor enabled sensitive cortisol detection with a linear range of 0.1 pM to 1 μM and a detection limit of 0.038 pM, with accurate quantification verified in human sweat and pharmaceutical samples. By integrating antifouling and regeneration, this work established a new strategy for robust and reusable MIP biosensors, addressing a key barrier to their practical application.