Glucose dehydrogenases (GluDH) from Bacillus species offer several advantages over other NAD(P)H regeneration systems including high stability, inexpensive substrate, thermodynamically favorable reaction and flexibility to regenerate both NADH and NADPH. In this research, characteristics of GluDH from Bacillus amyloliquefaciens SB5 (GluDH-BA) was reported for the first time. Despite a highly similar amino acid sequence when comparing with GluDH from Bacillus subtilis (GluDH-BS), GluDH-BA exhibited significantly higher specific activity (4.7-fold) and stability when pH was higher than 6. While an optimum activity of GluDH-BA was observed at a temperature of 50 °C, the enzyme was stable only up to 42 °C. GluDH-BA exhibited an extreme tolerance towards n-hexane and its respective alcohols. The productivity of GluDH obtained in this study (8.42 mg-GluDH/g-wet cells; 1035 U/g-wet cells) was among the highest productivity reported for recombinant E. coli. With its low K M-value towards glucose (5.5 mM) and NADP(+) (0.05 mM), GluDH-BA was highly suitable for in vivo applications. In this work, a recombinant solvent-tolerant B. subtilis BA overexpressing GluDH-BA was developed and evaluated by coupling with B. subtilis overexpressing an enzyme P450 BM3 F87V for a whole-cell hydroxylation of n-hexane. Significantly higher products obtained clearly proved that B. subtilis BA was an effective cofactor regenerator, a valuable asset for bioproduction of value-added chemicals.