Provided are a Moloney murine leukemia virus-based self-inactivating vector and applications thereof. The self-inactivating vector comprises: 5′LTR, a target expression gene or multiple cloning sites used for inserting the target expression gene, 3′LTR, and a polyadenylated nucleic acid fragment, where the nucleic acid fragment between the Pvu II enzyme cutting site and the Sac I enzyme cutting site of the U3 region of 5′LTR is replaced by a CMV enhancer and a CMV promoter connected to the CMV enhancer, the nucleic acid fragment between the Pvu II enzyme cutting site and the Sac I enzyme cutting site of the U3 region of 3′LTR is deleted, and the polyadenylated nucleic acid fragment is located at the 3′-terminus of 3′LTR. The self-inactivating vector, serving as a vector for a gene therapy, provides increased safeness and a high virus titer, and allows the high-efficiency expression of a target gene.