Because of the interference of maternal antibodies, the recombinant fowlpox virus (rFPV) vaccine has not been used widely. The selection of a well-defined FPV nonessential region might be a desirable way to solve this problem. Two pairs of primers were designed according to the genome of a pathogenic FPV to amplify two flanking regions (FPV1 and FPV2) of the supposed nonessential region by PCR, and then a series of plasmid vectors were constructed to generate the expression vector pP12LS, which containing FPV1, FPV2, the expression cassette of P11-LacZ reporter gene and the promoter Ps. To abtain the vector pP12LSF, the F gene of ZJ1 strain Newcastle Disease Virus (NDV) was inserted into pP12LS, in which the F gene was located downstream of the promoter Ps. pP12LSF was transfected into chicken embryo fibroblast (CEF) pre-nfected with 282E4 strain FPV. The recombinant FPV, rFPV-FSC, was purified by blue plaque selection. The LacZ and F genes could be expressed by rFPV-FSC after 20 successive passages in CEF. The FPV nonessential region was the only difference between rFPV-FSC and rFPV-FSB. These two rFPVs could induce completely protection in SPF chickens against lethal challenge with F48E8 strain NDV. However, the protective efficacy showed a significant difference in commercial chickens with maternal antibodies. The protective efficacy of rFPV-FSC was 100% and rFPV-FSB was 61.54%. The results indicate that the selection of a well-defined FPV nonessencial region is an effective way to increase the protective efficacy of rFPVs, especially in chickens with maternal antibodies.