The fermentation conditions were optimized by using a shaking bottle for the recombinant Escherichia coli BL21(DE3)/ pET28b-FDH exogenous expressions of formate dehydrogenase. The results showed that the soluble protein expression could reach 52.12 mg/g after induction by 0.1 mmol/L IPTG at 22 ℃ for 16 h, which was increased by 29.91% compared with that before optimization (40.12 mg/g). On this basis, the fermentation conditions of exogenous formate dehydrogenase were further optimized in 5 L fermenter. The optimized soluble protein expression in 5 L fermenter reached 41.26 mg/g after induction by 8 g/L lactose at 22 ℃ for 16 h. It increased by 20.82% compared with that before the optimization (34.15 mg/g). The obtained crude formate dehydrogenase from this fermentation together with threonine deaminase and leucine dehydrogenase were used as biocatalysts. Under the conditions of stirring speed 500 rpm, 35℃, 180 g/L L-threonine concentration, 120 g/L substrate ammonium formate, 0.1 g/L NAD+ in 1 L reaction system, after 9 h reaction, the substrate conversion rate was more than 99%, space-time yield was 17.2 g/(L·h), and e.e. value was more than 99.5%.