[Aim] With the increased number and variety of genetically modified (GM) plants and their derived products in the world, detection methods that can efficiently detect multiplex transformation vector simultaneously become increasingly important.[Method] Based on flanking sequences of five GM canola line (RF1, MS8, Topas19/2, Oxy235 and RF3) and endogenous reference gene cruciferinA (CruA) for canola, event-specific primers were designed. To analyze specificity and sensitivity of primers, GM canola lines, GM soybean lines, GM maize lines, GM rice lines and GM cotton lines were amplified by single PCR and different DNA concentrations of GM canola were amplified by multiplex PCR.[Result] The developed 6×multiplex PCR assay could specifically detect five canola lines, RF1, MS8, Topas19/2, Oxy235 and RF3. The sensitivity of multiplex PCR detection was 0.05%.[Conclusion] The developed 6×multiplex PCR system has high efficiency, specificity and sensitivity, and could meet the requirements of transgenic component detection. All these results suggested the developed multiplex PCR system could be applied in GM canola detection.