In order to improve the expression level of Tobacco curly shoot virus (TbCSV) replication-associated protein gene (Rep) in Escherichia coli, the effects of various factors on the protein expression were studied, which included bacterial strain, culture temperature, inducing time, and the final concentration of inductor IPTG etc. The results indicated that the expression level of GST-Rep fusion protein reached highest using Rosetta as bacterial strain and induced with 0.5 mmol·L-1 IPTG as final concentration for 4 h at 28℃ after cultured for 3 h at 37℃, and the expression GST-Rep fusion protein accounted for 42% of the total cell lysates with the above culture conditions. The Rep fusion protein was purified with GST-Sepharose 4B affinity chromatography, SDS-PAGE analysis showed that the molecular mass of GST-Rep fusion protein was about 66 kD, and Western blot analysis indicated the GST polyclonal antibody could specifically bound to purified GST-Rep fusion protein. The optimized expression conditions of Rep will be useful for future research on function of Rep.