Abstract: To construct prokaryotic expression vector of thymosin alpha l (Tαl), and express in Escherichia coli strain BL21, make foundation to gain amounts of thymosin alpha l (Tαl). The four repeats of thymosin alpha l (Tαl), which was chemically synthesized, was inserted into expression vector pET32a. The recombined pET32a-3Tαl was transferred into Escherichia coli strain BL21with CaCl2. After screening with Ampicillin, the positive clone was induced to express with IPTG, and the expression product was analyzed with SDS-PAGE. A positive protein band, which was the same as interest protein molecular weight about 31kD, was found in the expression product. The gene of Tαl was cloned into the expression vector correctly and was expressed successfully in E. coli expression system.