Cell sheet technology has been widely employed for the treatment of myocardial infarction (MI), but cell sheet fabrication generally requires the use of thermo-responsive dishes. Here we developed a method for the preparation of adipose-derived stem cell (ASC) sheet that obviated the need of thermo-responsive dishes. This method only required the seeding of rabbit ASC onto 6-well plates at an appropriate cell density and culture in appropriate medium, and the cells were able to develop into ASC sheet in 2 days. The ASC sheet allowed for transduction with the hybrid baculovirus at efficiencies >97%, conferring robust and prolonged (>35 days) overexpression of vascular endothelial growth factor (VEGF). The ASC sheet was easily detached by brief (10 s) trypsinization and saline wash, while retaining the extracellular matrix and desired physical properties. The ASC sheet formation and VEGF expression promoted cell survival under hypoxia in vitro. Epicardial implantation of the VEGF-expressing ASC sheet to rabbit MI models reduced the infarct size and improved cardiac functions to non-diseased levels, as judged from the left ventrical ejection fraction/myocardial perfusion. The VEGF-expressing ASC sheet also effectively prevented myocardial wall thinning, suppressed myocardium fibrosis and enhanced blood vessel formation. These data implicated the potential of this method for the preparation of genetically engineered ASC sheet and future MI treatment.