NAD -dependent formate dehydrogenase (FDH) catalyzes the conversion of formate and NAD to produce carbon dioxide and NADH. The reaction is biotechnologically important because FDH is widely used for NADH regeneration in various enzymatic syntheses. However, major drawbacks of this versatile enzyme in industrial applications are its low activity, requiring its utilization in large amounts to achieve optimal process conditions. Here, FDH from Bacillus simplex (BsFDH) was characterized for its biochemical and catalytic properties in comparison to FDH from Pseudomonas sp. 101 (PsFDH), a commonly used FDH in various biocatalytic reactions. The data revealed that BsFDH possesses high formate oxidizing activity with a k value of 15.3 ± 1.9 s at 25°C compared to 7.7 ± 1.0 s for PsFDH. At the optimum temperature (60°C), BsFDH exhibited 6-fold greater activity than PsFDH. The BsFDH displayed higher pH stability and a superior tolerance toward sodium azide and H O inactivation, showing a 200-fold higher K value for azide inhibition and remaining stable in the presence of 0.5% H O compared to PsFDH. The application of BsFDH as a cofactor regeneration system for the detoxification of 4-nitrophenol by the reaction of HadA, which produced a H O byproduct was demonstrated. The biocatalytic cascades using BsFDH demonstrated a distinct superior conversion activity because the system tolerated H O well. Altogether, the data showed that BsFDH is a robust enzyme suitable for future application in industrial biotechnology.