Objective：To construct the recombinant adenovirus vector which can target EB virus positive lymphoma and express wild-type P53 gene. Methods：The rAd5.orip-CMV.P53 was generated by contransfection of 293 cells with pDC311.orip-CMV.P53.IRES-EGFP and the adenovirual packaging plasmid PBHGE3. The construction of virus was identified by PCR method, and the titer was measured by TCID50. To observe the specificity of the recombinant adenovirus, EBV-positive raji cells and EBV-negitive jurket cells were infected using rAd5.orip-CMV.P53 respectively. Results：The recombinant adenovirus targeting EBV-postive lymphoma and expressing P53 gene was successfully constructed. The results in vitro showed the recombinant adenovirus has specificity to EBV-positive lymphoma cells. Conclusion：rAd.orip-CMV.P53 may be used for targeting gene therapy of EBV-positive lymphoma.