Propionibacterium freudenreichii has been successfully applied to industrial anaerobic production of vitamin B-12. In the traditional fed-batch fermentation process, 5,6-dimethylbenzimidazole (DMB) is used as a precursor. However, DMB can inhibit the synthesis of vitamin B-12 metabolic intermediates, which is not conducive to semicontinuous fermentation of P. freudenreichii. To improve the fermentation efficiency, vitamin B-12 ex-situ biosynthesis was proposed and a membrane separation coupling fermentation technology was designed with adenosylcobinamide (Ado-cbi) as the indicator signal. The results showed that P. freudenreichii cells should be partly separated online at 84 h with 0.22-mu m spiral membrane. The fourfold concentrated suspension was suitable for ex-situ biosynthesis of vitamin B-12 with the addition of 3.60 mg L-1 DMB at 30 degrees C and pH 7.0. In the semi-continuous coupling fermentation process, vitamin B-12 concentration reached 56.76 +/- 3.86 mg L-1 , an improvement of 161.6 %, while reutilizing fermentation wastewater and improving the anaerobic fermentation efficiency of P. freudenreichii.