Soil microorganisms play important roles in the rhizosphere in various biological activities in natural and agricultural ecosystems. We performed a two-year field experiment that featured the cultivation of three Bt transgenic oilseed rape (Brassica napus L.) plant lines to evaluate their effects on rhizosphere microorganisms using phospholipid fatty acid (PLFA) profiling. A total of 27 fatty acids were detected in the rhizosphere soil of transgenic oilseed rape during the two consecutive planting years. During plant growth stages in the first year, there were no significant changes in the microbial community structure and diversity in rhizosphere soil samples, except a lower total fungi content (4.09 μmol g⁻¹) at the bolting stage. During the second year, the soil microbial community associated with transgenic plants varied significantly with developmental stage, such as total PLFAs (43.70 μmol·g⁻¹), PLFAs of bacteria (26.00 μmol g⁻¹) and fungi (11.84 μmol g⁻¹). The Shannon's diversity, richness, and evenness of the soil microbial community increased significantly with transgenic GT1 and GT5 lines during the flowering stage. PCA analysis of the PLFA profile explain 87.41% variation of PLFAs in 2013 and 86.96% in 2014, showing that the different transgenic lines might have a minor impact on soil microbial structure. This study demonstrated that field cultivation of transgenic oilseed rape may cause changes in soil microbial structure and diversity for consecutive seasons and at different growth stages.