AbstractThere is increasing demand for direct in situ metabolite monitoring from cell cultures and in vivo using implantable devices. Electrochemical biosensors are commonly preferred due to their low‐cost, high sensitivity, and low complexity. Metabolite detection, however, in cultured cells or sensitive tissue is rarely shown. Commonly, glucose sensing occurs indirectly by measuring the concentration of hydrogen peroxide, which is a by‐product of the conversion of glucose by glucose oxidase. However, continuous production of hydrogen peroxide in cell media with high glucose is toxic to adjacent cells or tissue. This challenge is overcome through a novel, stacked enzyme configuration. A primary enzyme is used to provide analyte sensitivity, along with a secondary enzyme which converts H2O2 back to O2. The secondary enzyme is functionalized as the outermost layer of the device. Thus, production of H2O2 remains local to the sensor and its concentration in the extracellular environment does not increase. This “biostack” is integrated with organic electrochemical transistors to demonstrate sensors that monitor glucose concentration in cell cultures in situ. The “biostack” renders the sensors nontoxic for cells and provides highly sensitive and stable detection of metabolites.