We aimed to control the gene expression of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in the ischemic heart to explore the feasibility of sequential, timely and controlled multigene expression as a means of improving therapeutic angiogenesis in vivo. Adult rabbit myocardial infarction models were surgically established (n = 120). Hypoxia-inducible factor-1α-hypoxic response element (HIF1α-HRE) and Tet (tetracycline)-On advanced gene control systems were reconstructed for controlled expression of the human VEGF165 (hVEGF165) and Ang-1 genes, respectively. Recombinant adeno-associated viruses (rAAV)-9HRE-hVEGF165 and rAAV-TRE-Tight-Ang-1 were delivered into the ischemic myocardium for 12 weeks. Reverse transcription-polymerase chain reaction, western blotting and immunofluorescence staining were used to detect gene and protein expression. Vessel functionality, vascular permeability and animal cardiac function were also evaluated. Under the control of the HIF1α-HRE and Tet-On gene control systems, the expression of the exogenous hVEGF165 and Ang-1 genes was consistent in the ischemia control. In the sequential group, we found that the number of functional vessels with a larger diameter and more vascular branches was increased, and vascular permeability was significantly reduced. In addition, animal heart function was significantly improved compared with the non-sequential and hVEGF165- or Ang-1-only groups (P<0.05, P<0.05, respectively). Sequential, timely and controlled expression of the hVEGF165 and Ang-1 genes in vivo is a new therapeutic angiogenesis strategy that can effectively promote functional vessel regeneration and can improve cardiac function in ischemic heart disease.