By optimizing different parameters for infection, an efficient Agrobacterium-mediated transformation system for Brassica napus was developed. A series of combinations of optical densities, infection durations, concentrations of acetosyringone and silwet L77 were evaluated. Maximum transformation efficiency reached 18.93% when cotyledonary nodes were infected for 10 min with an Agrobacterium suspension of 0.8 OD and 200 mu M acetosyingone and 0.02% silwet L77 in the culture medium. In addition, the concentrations of 6-benzyladenine and naphthalene acetic acid were also optimized. Meanwhile, maximum transformation efficiency for green plantlet was 94.38% when cotyledonary nodes were cultivated in the medium containing 4 mg/L 6-benzyladenine and 0.04 mg/L naphthalene acetic after co-cultivation. Transformation was confirmed by polymerase chain reaction analysis, southern blotting analysis and green fluorescent protein assay.