Enterotoxigenic Escherichia coli (ETEC) is an important pathogen causing diarrheal disease in humans and animals in developing countries. EtpA adhesin and heat-labile enterotoxin B subunit (LT B) are the main virulence factors of ETEC. The purpose of this study is to construct a prokaryotic expression vector for EtpA and LT B and to purify these two  proteins. The cloning of the EtpA and LT B genes from ETEC using polymerase chain reaction (PCR) was performed. The fragments were then identified and cloned into the prokaryotic expression vector pET-32a. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express with IPTG. The products of expression were analyzed by SDS-PAGE and western-blot. We obtained the optimum expression conditions of pET-EtpA and pET-LT B. The maximal expression quantity of EtpA was observed at 5 h of induction by 1 mM IPTG at 37 °C, and, whereas that of LT B was observed at 6 h of induction by 1 mM IPTG at 37 °C. The expression of EtpA and LT B provides the material basis for the further development of novel ETEC vaccine.