The substitution of the rFVIII sequences in hFVIII led to an increased coagulation activity. The expression plasmids of HC (CB-hHC, CB-rHC) were co-transfected into 293 T cells with LC (CB-hLC, CB-rLC). The total amount of the plasmids of each group was 1 μg and the ratios of HC:LC were 1:1 and 2:1. The activity of FVIII (hHC +hLC, hHC +rLC, rHC +hLC, rHC +rLC) was detected at 48 h post-transfection by the aPTT assay. NHP (normal human plasms) was used as the standard (n =3, *p < 0.05, ****p < 0.0001).