Abstract: LrrG (leucine-rich repeat protein from GBS) and Sip (surface immunogenic protein), which are two kinds of surface antigen proteins from Streptococcus agalactiae in tilapia, have good immunogenicity. To obtain the LrrG-Sip fusion protein via coalescing surface antigen protein LrrG and Sip of S.agalctiae in tilapia, we cloned Sip and LrrG genes into vector pCold Ⅱ one by one using double enzyme method of gene splicing technology, and constructed a prokaryotic expression vector pCold Ⅱ-LrrG-Sip. The recombinant plasmid was transformed into E.coli BL21(DE3), and the result indicated that 9 h, 15 ℃, 0.5 mmol·L^-1 IPTG were the optimum inducing conditions under which fusion protein was most soluble and abundant. Western blotting test showed that the LrrG-Sip fusion protein was about 160 kDa, consistent with the prediction (162 kDa), which suggested the prokaryotic expression vector pCold Ⅱ-LrrG-Sip was constructed successfully and laid the foundation for developing subunit vaccines for S.agalctiae in tilapia.