In order to construct an efficient and safe engineering vaccine against E. tenella, the relative gene of Rhomboid protein family of E. tenella F2 hybrid strain was amplified by RT-PCR technology and subcloned into the downstream of complex promoter of fowlpox virus vector pUTA2, recombinant fowlpox virus transfer vector was constructed successfully. Plasmid of fowlpox recombinant virus and fowlpox virus were co-transfected into chicken embryo fibroblast (CEF) and identified by BrdU drug, RT-PCR and Western blotting respectively. The result showed that the E. tenella fowlpox recombinant virus (rFPV-Rhomboid) was obtained. Furthermore, rFPV-Rhomboid was amplified in CEF, then chicken was immuned with rFPV-Rhomboid and the indication of immunity was detected. The result indicated that the contents of CD4⁺ and CD8⁺ in peripheral blood were significantly higher than that in control group (P<0.05). The weight gained of chicken vaccinated rFPV-Rhomboid was different from the chickens of control group significantly (P < 0.05). The recombinant protein had immunogenicity and could induced strong immune response and immune protection ability.