Objective To establish a green fluorescent protein (GFP) / firefly luciferase (Luc) double-labeled ECA109 human esophageal cancer cells (ECA109-Luc-GFP) and apply them to a mouse model of esophageal cancer for non-invasive bioimaging. Methods MTT, flow cytometry (FCM), and wound healing assays were used to analyze biological differences between ECA109-Luc-GFP cells and parental ECA109 cells. Esophageal cancer xenografts were established by subcutaneous or tail vein injections of ECA109-Luc-GFP cells into nude mice. An in vivo imaging system was used to observe the growth of xenografts. Results MTT, FCM, and wound healing assays showed no differences between ECA109-Luc-GFP and parental cells in terms of cell viability, proliferation, and metastasis ( P > 0. 05). The growth and metastasis of xenografts established by ECA109-Luc-GFP cells were observed using the in vivo imaging system.Conclusions A GFP/ Luc double-labeled human esophageal cancer cell line is established, and the growth and metastasis of xenografts can be observed by an in vivo imaging system. This study provides the experimental basis for an esophageal cancer mouse model and clinical study.