Objective To construct a baculovirus expression vector for expression of human Ro60 cDNA. Methods Ro60 eDNA was cloned from Hela cell RNA with RT-PCR and was inserted into plasmid pFastBac HTc. Plasmid pFastBac HTc-Ro60 was then transformed into E. coli, DH10Bac competent cells for transposition. The constructed recombinant was determined by both blue-white colony screening and PCR analysis. Results A 1.56kb Ro60 eDNA was obtained from Hela cell RNA and was successfully inserted to plasmid pFastBac HTc. DNA sequencing and restriction enzyme analysis revealed that both open reading frame and sequence of the cDNA were identical to Ro60 sequence previously published. Specific transposition and virus recombination were obtained in the vector Bacmid-Ro60. Conclusion Ro60 eDNA has been successfully cloned and constructed to baculovirus expression vector, which is useful for protein expression and further study of this autoantigen in the pathogenesis of lupus erythematosus.