Co-delivery of B-domain-deleted FⅧ(BDD-FⅧ) heavy and light chain genes by dual-vector system is one of strategies to overcome packaging constraint of adeno-associated virus vectors (AAV), but leading to chain imbalance because of inefficient secretion of heavy chain. We hypothesize that a disulfide linking between the heavy and light chains can improve the efficacy of dual-vector co-delivery of BDD-FⅧ gene. Here, we plan to achieve this by making Cys mutations at the Met662 in A2-domain of heavy chain and Asp1828 in A3-domain of light chain. By co-transfecting cultured COS-7 cells transiently with mutant heavy and light chain genes, a disulfide cross-linked heavy and light chain was observed by Western blotting. The secretion of heavy chain and coagulation activity were up to (109±13) μg/L and (0.65±0.12) U/ml respectively analyzed by ELISA and Coatest method greater than that of two chains of wild-type BDD-FⅧ gene co-transfected cells((71±11) μg/L and (0.35±0.05) U/ml. It suggests that inter-chain disulfide linking could efficiently improve efficacy of dual-vector delivery of the BDD-FⅧ gene. It provided evidence for ongoing in vivo BDD-FⅧ gene delivery by dual-AAV vector.