Objective To construct novel recombinant baculoviruses with Tet-On system and enhanced green fluorescent protein (EGFP) or hepatic growth factor (HGF) which could be regulated by different concentrations of doxycycline (DOX). Methods The recombinant plasmids pFast-Tet, pTRE-EGFP and pTRE-HGF were digested. The target fragments were collected and connected to pFast-Tet, the resultants were used to transform DH10Bac competent cells containing AcMNPV Bacmid and helper plasmid, and the Bacmid DNA were identified (named Ac-EGFP and Ac-HGF) after selection and extraction. Ac-EGFP and Ac-HGF were then transfected into bone mesenchymal stem cells (BMSCs), and the expression of EGFP and HGF were regulated by different concentrations of DOX (EGFP:0, 200, 500, and 1 000 ng/mL; HGF: 0, 10, 100, 500, 1 000, and 1 200 ng/mL); EGFP expression was observed under fluorescence microscope and the level of HGF expression was detected by ELISA. Results It was verified that Tet-On system was successfully constructed in a baculovirus vector with EGFP or HGF, and they were highly transfected into BMSCs. EGFP and HGF were highly expressed when exposed to high concentrations of DOX. And the expression of EGFP and HGF were gradually decreased at low concentration or absence of DOX. Conclusion Tet-On system can be used to construct a new recombinant baculovirus vector containing EGFP or HGF and it can stably and highly transfect BMSCs; different concentrations of DOX can lead to different expression of EGFP and HGF, and they are in low background expression without DOX.