The autophagy is an important metabolic process. In order to better study autophagy under different conditions, an autophagy detection plasmid pLVX-MRFP-EGFP-LC3 was constructed. The total RNA of Hela cells was extracted by Trizol. LC3 fragment was specifically amplified by PCR technique, and then inserted into the plasmid pEGFP-N3 vector. The mRFP fragment was inserted into the EGFP-LC3 plasmid, and the mRFP-EGFP-LC3 fragment with different restriction sites was amplified by PCR and inserted into the lentiviral vector pLVX-puro. The plasmid was transiently transfected into 293T cells with Fugene?6. The expression of the fluorescent protein was detected by fluorescence microscope. The expression of the target gene was detected by western blotting. The results showed that the autophagy detection plasmid pLVX-mRFP-EGFP-LC3 was successfully constructed. Red and green fluorescence were observed under fluorescence microscope. The expression of LC3 was detected by western blotting. This study laid the foundation for further study of autophagy.