Organic photoelectrochemical transistors (OPECT) have emerged as leading candidates for biosensing technologies, attributed to their high transconductance, zero operating voltage, and excellent biocompatibility. However, the practical application of OPECT in complex biological fluids is usually hindered by challenges such as complex gate structures, undesired contamination, and side reactions. Herein, a contamination‐free OPECT biosensor is proposed based on a nanochannel membrane‐isolated design, effectively preventing the biological matrix from the detection cell. Using adenosine triphosphate (ATP), a biomarker for malignant tumors and Alzheimer's disease for proof‐of‐principle, the metal−organic framework (MOF)‐guarded nanochannel entrance is switch‐on by the strong chelation interaction between ATP and the Zn(II) nodes in MOFs, which allowed sodium thiophosphate substrate to pass through. Benefiting from the confinement effect of nanochannels and frameworks, the encapsulated alkaline phosphatase molecules exhibited high catalytic efficiency for H2S production and subsequent CdS production on gate electrode, thus altering the transistor response. The OPECT device, combined with the nanochannels‐regulated on‐off strategy not only effectively prevented contamination of OPECT from complex biomatrix but also enabled rapid and direct detection of 1.0 nm ATP within 15 min, providing an alternative insight into the design of OPECT device for interference‐free rapid detection in biological matrices.