In order to improve the Agrobacterium tumefaciensmediated transient expression system in grapevine callus, we used the callus of grape ‘Thompson Seedless’, which was induced from stem segment with single bud, to analyze some major factors influencing the transient expression efficiency, such as the pH of A. tumefaciens suspension, the concentration of A. tumefaciens suspension and the time of vacuum infiltration. The results showed that: (1) the best media for the induction of grapevine callus were hormone combination series of 1.0 mg/L BAP，1.0 mg/L BAP+0.02 mg/L NAA，2.0 mg/L BAP+0.02 mg/L NAA and 4.0 mg/L BAP+0.02 mg/L NAA, comparing with the hormone combination which is added 0.1 mg/L 2, 4D or 0.1 mg/L TDZ. (2) The optimal combination was that the pH of A. tumefaciens suspension, the OD₆₀₀ of A. tumefaciens suspension and the time of vacuum infiltration were 5.1, 1.0 and 20 min, respectively. (3)The optimized transient expression system had significant influence on the transient expression efficiency in different organs of Thompson Seedless. The transient expression efficiency of callus is the significantly highest (6 5231.99±3 339.29 mU/g), and the GUS staining result of callus is the darkest. However, the transient expression efficiency of leaves is the lowest. The transient expression vector pCAMBIA0390∷GUS with report gene coding βDglucuronidase (GUS) was transferred into callus by A. tumefaciens mediated transformation. The results of GUS activity proved that the improved transient expression system in callus of grapes contributed to the expression of exogenous genes in the callus of grapes, which provides technical support for the study of gene function of grapevine.