We studied the purification of diacetyl reductase, the preparation of a biosensor, and its performance. With diacetyl reductase and reduced coenzyme I (NADH) co-immobilized as working membrane, NAD+/NADH produced in the course of diacetyl reduction was connected with Fe2+/Fe to build a biosensor. The biosensor could be used to determine diacetyl concentration within the range from 0.1 microgram/mL to 0.5 microgram/mL and the response time was less than two minutes, and its performance was stable within 9 days. The experiments showed that typical metal ions and organics in nominal concentration did not affect the performance of the biosensor, meanwhile, the interference of dissolved oxygen on the performance of biosensor and regeneration of coenzyme I (NADH) were solved to some extent.