Formate dehydrogenases are critical tools for nicotinamide cofactor regeneration, but their limited catalytic efficiency (k/K) is a major drawback. A formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) was the first native NADP-dependent formate dehydrogenase reported and has the highest k/K toward NADP (k/K) compared with other FDHs that can utilize NADP as a hydrogen acceptor. However, the substrate and cofactor affinities of BstFDH are inferior to those of other FDHs, making its practical application difficult. Herein, we engineered recombinant BstFDH to enhance its HCOO and NADP affinities. Based on sequence information analysis and homologous modeling results, I124, G146, S262, and A287 were found to affect the binding affinity for HCOO and NADP. By combining these mutations, we identified a BstFDH variant (G146M/A287G) that reduced K to 0.09?mM, with a concomitant decrease in K, and gave 1.6-fold higher k/K than the wild type (WT). Furthermore, BstFDH I124V/G146H/A287G, with the lowest K of 8.51?mM, showed a catalytic efficiency that was 2.3-fold higher than that of the wild type and a decreased K of 0.11?mM. These results are beneficial for improving the performance of NADP-dependent formate dehydrogenase in the NADPH regeneration of various bioreductive reactions and provide a useful guide for engineering of the substrate and cofactor affinity of other enzymes.