We constructed a recombinant strain BL21 (DE3) (pETST3LTBalphabeta) including ST1-LT(B)-alpha-beta fusion gene via molecular technology. The SDS-PAGE and Western blotting indicated that the ST1-LT(B)-alpha-beta fusion protein was highly expressed in Escherichia coli and the molecular weight of the fusion protein was about 110 kD. The recombinant strain was induced in different concentrations of lactose and different aeration rate. The optimal culture conditions in 20 L fermentor were 1% inoculation (V/V), initial aeration 5 L/min, 0.03 mol/L lactose addition 3 hours after inoculation, and increased the aeration to 12.5 L/min for the following 6 hours. The fusion protein was about 38.53% of total cellular protein. It was nontoxic, immunogenic and protective against enterotoxigenic E. coli and Clostridium perfringens infection. The constructed recombinant strain BL21 (DE3) (pETST3LTBalphabeta) could serve as a candidate vaccine strain against diarrhea of piglet.