The LuxS/AI-2 quorum sensing (QS) system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). In this study, the method of biosynthesis of AI-2 was established using recombinant LuxS and Pfs of avian pathogenic Escherichia coli (APEC), which will be benefit for future study of the role of AI-2 in APEC. We investigated AI-2 production in APEC by vibrio harveyi BB170 (BB170). Furthermore, APEC LuxS and Pfs were expressed, purified and used to investigate the production of AI-2 in vitro. LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH), the reaction was detected for the production of luminescence of BB170. APEC can produce AI-2 by BB170 bioassay. Purified LuxS and Pfs enzymes incubated with SAH and produced 300 micromol/L AI-2 in the reaction products. The results demonstrated that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH. These findings will be of benefit to future studies of the role of AI-2 in APEC.