Objective To construct the recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene and provide the gene therapic strategy for hepatocellular carcinoma. Methods The pAdTrack-EAFP-PALB was constructed and the r-Caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdEasy-EAFP-PALB/r-Caspase-3 vector was digested with PacⅠand transfected into AD293 cells for packaging and amplifying, recombinant virus was constructed successfully. Infection titer and efficiency of recombinant virus were monitored by green fluorescent protein (GFP) expression. The expression of r-Caspase-3 in infected HepG2 cells was detected by RT-PCR and Western blot. The apoptosis of HepG2 cells was detected by SRB dyeing method. Results Shuttle vector pAdTrack-EAFP-PALB/r-Caspase-3 was correct after identification by restriction endonuclease analysis and sequencing. By PCR and PacⅠ restriction endonuclease analysis, the homologous recombinant of pAdEasy-EAFP-PALB/r-Caspase-3 was successful. The expression of GFP was observed when linearized pAdEasy-EAFP-PALB/r-Caspase-3 was transfected into AD293 cells. AD293 cells could be infected repeatedly by recombinant adenovirus. The expression of r-Caspase-3 gene on HepG2 cells was detected by RT-PCR and Western-blot methods respectively, which confirmed that the Ad-EAFP-PALB/r-Caspase-3 was constructed successfully. The specificity of Ad-EAFP-PALB/r-caspase-3 which targeting induced hepatocellular carcinoma cells was founded by SRB dyeing test. Conclusion The Recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene was constructed successfully and which established the foundation of r-Caspase-3 gene therapy in future research to hepatocellular carcinoma.