After two retroviral systems with enhanced green fluorescent protein (EGFP) report gene were constructed in this study, the retroviral systems and the infection method suitable for buffalo fetal fibroblasts (BFFs) were investigated. The results revealed that the retrovirus titer of the two systems was 10⁶, and the titer of the pMX system could be up to 10⁷ after ultracentrifugation concentration. The infection efficiency of pMX retroviral system was higher than that of the pMSCV retroviral system. Using the fresh retrovirus infected BFFs twice (12 h interval) or ultracentrifugal retrovirus infected BFFs once could improve the infection efficiency.