Abstract: NAD+ depended formate dehydrogenase (FateDH) from Candida boidinii was purified by diethylaminoethyl (DEAE) Sepharoses Fast Flow chromatography after the strain was cultured by two-step fermentation and crude cell extract was obtained by crushing and separation. The enzyme specific activity increased from 0.83 U/mg to 2.67 U/mg and a 3.22 fold purification was achieved with the recovery of 29.7% activity. The ordered BiBi reaction mechanism of FateDH enzymatic reaction was confirmed by the study of possible course from reactant consumption to product production. NAD+ was the first substrate with HCOONa as the second substrate. NADH was the first release product with HCO3ˉ as the subsequent release second product. The kinetics parameter including Vmax, KiS1, KmS2, KmS1 were solved by quadratic drawing and the BiBi reaction kinetic model of FateDH was established as .