Glufosinate is a non-selective broad-spectrum herbicide applicable in weeds control worldwide. However, in China there is no glufosinate-tolerant rapeseed variety with independent intellectual property rights to date. In this study, a glufosinate-resistant gene Syn1-RePAT was successfully transformed to the Brassica napus via Agrobacterium tumefaciens-mediated hypocotyl method, and 27 transgenic plants were obtained. The transgenic lines with a single copy of T-DNA insertion were identified by Southern blot analysis, and some of them on T-DNA copy number variation were analyzed by progeny segregation assay. The T-DNA insertion sites in the lines of OV40-7, OV40-15, OV40-16 and OV40-17 were identified by inverse PCR method. The stability of the T-DNA insertion was further confirmed by insertion-specific PCR in their T1 to T3 generations. RT-PCR and qRT-PCR expression analysis revealed that the Syn1-RePAT gene was stably overexpressed in different generations of transgenic lines. Treatments with different doses of glufosinate indicated that lines OV40-6, OV40-7 and OV40-16 were tolerant to at least six times of the recommended dose of glufosinate in production. Thus, the novel glufosinate -tolerant rapeseed lines generated in the present study will lay the foundation for the herbicide- tolerance rapeseed breeding in China.