Objective To explore the feasibility of in vivo tracking of adipose-derived stem cells (ADSCs) by using lentiviral vectors containing green fluorescent protein (GFP) and luciferase (Luc) dual reporter gene transfection and bioluminescent imaging techniques. Methods The ADSCs were isolated from human adipose tissue samples of cosmetic subdermal liposuction. The ADSCs were transfected by lentiviral vectors containing GFP and Luc. The labeling efficiency was evaluated by fluorescence microscope and flow cytometry in vitro. The transfected ADSCs were subcutaneously injected into BALB/C mice, and then a non-invasive bioluminescence imaging procedure was performed 1 h, 1, 2, 4, 6 and 8 weeks after transplantation. The DAPI staining of skin tissues was performed to identify the survival and the location of transplanted ADSCs. Results High GFP expression was observed in transfected ADSCs by lentiviral vector under fluorescence microscope. The transfection efficiency of lentiviral vectors containing GFP and Luc was 87.3%. Transfected ADSCs can express GFP and Luc after 25 successive generations, and the transduction of ADSCs with lentiviral bioluminescence reporter vectors had no effect on cell proliferation. The bioluminescence of ADSCs in vivo can be detectable as long as 8 weeks after transplantion, and the ADSCs mainly survive in the inoculating site of the thigh. Histological analysis of sections showed that the labeled ADSCs located in the subcutaneous layer of the implantation site. Conclusion Using lentiviral vectors containing GFP and Luc dual reporter gene transfection and bioluminescent imaging techniques can monitor ADSCs in vivo.