Vitis vinifera cv. Chardonnay suspension cultures were established from proembryogenic mass and employed for optimizing Agrobacterium-mediated transformation system. One-factor-at-a-time experiment revealed that OD600 of Agrobacterium,time of inoculation, co-cultivation, and cell-drying before inoculation significantly affected the transformation efficiencywhich reached maximum 21.5% at the following conditions: 0.8 of OD600, 25 min of inoculation, 2 d of co-cultivation, and 10min of cell drying. Response surface methodology experiments based on a five-level, four-factor central-compositerotatable design were then used to optimize these selected factors. The optimized conditions for Chardonnay grapetransformation were: 0.8711 of OD600, 28.9 min of inoculation, 2.25 d of co-cultivation and 11.76 min of cell drying. Afteroptimization, transformation efficiency was 26.2% and there were no interactions among different factors.