Purpose <p>High-throughput screening methods for cytochrome P450 enzymes (P450s), such as colorimetric, mass spectrometric, and fluorescence-based assays, often face limitations in throughput, real-time monitoring, and versatility.</p> Methods <p>To address these challenges, we developed a novel biosensor leveraging glucose-6-phosphate dehydrogenase and Bimolecular Fluorescence Complementation for real-time monitoring of intracellular NADP⁺ levels, enabling P450 activity detection. The sensor was applied to monitor P450 activity by tracking intracellular NADP⁺ dynamics, as P450s catalyze diverse substrate reactions and convert NADPH to NADP⁺ via their electron transport system. To enhance detection precision, intracellular NADP⁺ synthesis was reduced by knocking down NADPH-dependent aldehyde reductase (YqhD), minimizing background fluorescence interference.</p> Results <p>The sensor exhibited a linear NADP⁺ detection range of 1 μM to 10 mM, suitable for P450 assays. The sensor's performance was validated by comparing P450 activities in engineered strains with traditional gas chromatography.</p> Conclusion <p>The developed biosensor demonstrates its potential as a robust, real-time screening tool for P450 enzyme studies.</p>