Objective:To establish a stable GFP-LC3-expressed RAW264.7 cell line. Methods:The pcDNA3.1-GFP-LC3 plasmid was constructed and transfected into RAW264.7 cell with transfection reagent. The stable transfectants were screened by G418. The GFP-LC3 protein expression was analyzed by Western blot. The fluorescent signals were detected by inverted fluorescence microscope. ER stress-induced autophagy was detected by confocal microscope and Western blot. Results:Selected by G418,2 transfected cell lines showed high expression level of GFP-LC3,as demonstrated by Western blot analysis. More than 95% cells showed positive fluorescent signals under inverted fluorescence microscope. The formation of autophagosomes and the increases in the conversion of LC3-Ⅰ to LC3-Ⅱ was observed in the constructed cells when treated with the ER stress inducer,thapsigargin. Conclusion:A RAW264.7 cell line stably expressing GFP-LC3 was constructed successfully in the study.