Object With the development of the stem cell, the study of rat stem cell became urgent. The purpose of this study was to generate EGFP transgenic rat models for fluorescence imaging, tracking of stem cells homing and cell transplantation. Methods The enhanced green fluorescent protein (EGFP) transgenic rats were obtained by microinjection and the expression of EGFP was determined under in vivo fluorescence imaging system and laser scanning confocal microscope. The expression of EGFP in blood cells, bone marrow cells and bone marrow stem cells was investigated with flow cytometry. Results The EGFP transgenic rat SD-TgN(CAG-EGFP-1)ZLFILAS was established. EGFP was systematically expressed in tissues of heart, liver, muscle, lung, pancreas, brain, bladder, stomach, kidney, intestines and spleen, and EGFP labeling ratios in blood cells and bone marrow stem cells were 94.4% and 97.8% respectively. Conclusions A systematic EGFP transgenic rat line was generated which showed EGFP labeling ratios higher than 95% in bone marrow stem cells. This transgenic rat may be a useful rat tool for fluorescence imaging and stem cells tracking.