Abstract: To address the challenges of regioselective oxidation at the C2 position and precise control of stereochemistry at the C5 position in the total synthesis of Erythrina alkaloids, a relay oxidation system comprising P450 BM3 and SjAKR was constructed. However, the distinct cofactor requirements of P450 BM3 (for NADPH) and SjAKR (for NADP⁺) posed a bottleneck for the enzymatic synthesis of chiral intermediates. To achieve cofactor regeneration in this relay oxidation system, this study systematically evaluated three cofactor regeneration strategies: self-recycling, competitive, and independent formats. Results indicated that constructing independent cofactor regeneration systems for each enzymatic step was the most efficient approach. Among the enzymes tested, BstFDH_G164M/A287G was identified as the optimal NADPH-regenerating enzyme for the P450 BM3 catalytic cycle, while SmNOX was the best NADP⁺-regenerating enzyme to support the kinetic resolution catalyzed by SjAKR. After optimizing the reaction conditions, a "one-pot" enzymatic reaction was performed. The process involved an initial hydroxylation catalyzed by the P450 BM3/BstFDH_G164M/A287G system, followed by kinetic resolution via the SjAKR/SmNOX system. This strategy afforded the chiral intermediates (2R, 5S)-3 and (5R)-4 with isolated yields of 32% and 28%, and optical purities of 99% ee and 97% ee, respectively. Through systematic optimization of cofactor regeneration, this study achieved the efficient and highly selective synthesis of chiral intermediates, providing crucial technical support for the chemoenzymatic total synthesis of Erythrina alkaloids.