Though great advances are achieved in the area of organic electrochemical transistor (OECT) biosensors, general issues of shared electrolyte by the channel and gate electrode remain unsolved. On the other hand, to date binding‐free OECT detection of cellular membrane proteins is not reported. On the basis of the advanced technique of organic photoelectrochemical transistor (OPECT), herein the H‐cell‐supported OPECT device that can easily circumvent the issues of abovementioned shared electrolyte and its application toward binding‐free detection of membrane alkaline phosphatase (ALP) of HeLa cells is demonstrated. In such a configuration, usage of a Nafion perfluorinated membrane equipped H‐cell can well retain the ionic circuit of the device but prevent the diffusion of various molecules and minimize the possible interference. In the detection, ALP catalytic chemistry enables the sensitization of the TiO2 nanotubes electrode, triggering enhanced de‐doping of the poly(3,4‐ethylenedioxythiophene):poly(styrene sulfonate) and thus resulting in altered channel currents proportionate to the membrane ALP. This work not only provides a new perspective for binding‐free detection of membrane protein but also represents a general protocol for H‐cell‐supported OECT/OPECT biosensors with enhanced accuracy.