Objective Attenuated strain TTVAC7 is a multi-gene-deleted vaccinia virus Tiantan strain constructed by knocking out parts of non-essential genes related to the virulence, host range and immunomodulatory of vaccinia virus Tiantan strain (VTT), and combining the double markers screening and exogenous selectable marker knockout techniques.Methods In this study, shuttle vector plasmids pTC-EGFP, pTA35-EGFP, pTA66-EGFP, pTE-EGFP, pTB-EGFP, pTI-EGFP and pTJ-EGFP were constructed, which contained seven pairs of recombinant arms, and bound to early and late strong composed promoter pE/L, and to enhanced green fluorescent protein (EGFP) as an exogenous selectable marker.BH-K cells were co-transfected/infected successively with the above plasmids and VTT or gene-deleted VTT, and homologous recombination and fluorescence plaque screening methods were used to knock out the gene fragments (TC: TC7L ~ TK2L;TA35: TA35L;TA66: TA66R;TE: TE3L;TB: TB13R ~ TB14R;TI: TI4L;TJ: TJ2R).Cre / Loxp system was applied to knock out the exogenous selectable marker.A series of in vivo and in vitro experiments were used to detect virulence of TTVAC7.Results Ultimately, a gene-deleted attenuated strain TTVAC7 was obtained and A series of in vivo and in vitro experiments demonstrated that not only the host range of TTVAC7 could be narrowed and its toxicity might be weakened significantly, but also its high immunogenicity was maintained at the same time.Conclusion TTVAC7 shows a more excellent safety and immunogenicity than the wild virus, and a potential to be developed into a new virus vector vaccine for the prevention or treatment of diseases caused by different pathogens.