The invention discloses a CAR-T transgene vector based on replication defective recombinant lentivirus. The CAR-T transgene vector comprises an original nuclear replicon pUCOri sequence, a resistance gene AmpR sequence containing ampicillin, a virus replicon SV40 Ori sequence, a lentivirus packaging cis element, ZsGreen1 green fluorescent protein, an IRES ribosome binding sequence, a human EF1 alpha promoter , a chimeric antigen receptor of second-generation CAR or third-generation CAR and a regulating element, wherein the original nuclear replicon pUCOri sequence is used for plasmid replication; the resistance gene AmpR sequence is used for massively proliferating target strains; the virus replicon SV40 Ori sequence is used for enhancing replication in eukaryocyte; the lentivirus packaging cis element is used for lentivirus packaging; the ZsGreen1 green fluorescent protein is used for expressing green fluorescent for eukaryocyte; the IRES ribosome binding sequence is used for jointly transcribing and expressing protein; the human EF1 alpha promoter is used for conducting eukaryotic transcription on antigen receptor genes; the chimeric antigen receptor is used for forming the second-generation CAR or the third-generation CAR integrating recognition, transfer and start; the regulating element is used for enhancing expression efficiency of transgenes and used after eWPRE-enhanced type woodchuck hepatitis b virus is transcribed. Besides, the invention further discloses a construction method and application of the vector. By means of the CAR-T transgene vector and the construction method and application of the vector, secretion of cell factors and an in vitro killing effect of CAR-T cells can be remarkably improved, and the clinical treatment effect is remarkable.