Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC+ rLC protein had a higher activity than the hHC+ hLC protein at comparable expression levels. The specific activity of hHC+ rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC+ rLC treatment exhibited a visibly higher activity than those treated with hHC+ hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC+ rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy.