Aim To prepare catalase-contained recombinant adenovirus. Methods Catalase gene was digested from plasmid of pZeoSV2-Cat, subcloned into plasmid of pBluescript Ⅱ sk( + ) and formed plasmid of pBluescript Ⅱ sk( + )-Cat. Then Catalase gene was digested from plasmid of pBluescript Ⅱ sk( + ) -Cat, subcloned into shuttle plasmid of pShuttle-CMV and formed transfer plasmid of pShuttle-CMV-Cat. Adenovirus genomic plasmid of pAdEasy-1 was transformed into BJ5183 bacteria and prepared ultracompletent BJ5183 containing pAdEasy-1. pShuttle-CMV-Cat was linealinzed with Pme I and transformed into ultracompletent BJ5183 containing pAdEasy-1. The identified recombinant adenovirus plasmid of pAdEasy-1-Cat was linealinzed with Pac I and transfected into Ad-293 cells to package recombinant adenovirus particles. The target gene was decteted by PCR. Results The hneahnzed pShuttle-CMV-Cat was transformed into ultracompletent BJ5183 containing pAdEasy-1 .There were 30% positive recombinant plasmid. After pAdEasy-1-Cat was transfected into Ad-293 cells, recombinant adenovirus particles were produced and further amplified. PCR test indicated that the recombinant adenovirus AdCat contained Catalase gene. The liter of the purified AdCat was 4.12 × 1015 OPU/L. Conclusion The homologous recombination in bacteria is a convenient and high efficient method to prepare AdCat. This affords a good gene transfer vector for the gene therapy in restenosis.