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Rapeseed (Brassica napus L.) is an important oil crop worldwide. For current B. napus production, it is urgent to develop new varieties with higher seed productivity and increased stress tolerance for better adaptation to the abiotic stresses as a result of global climate change. Genetic engineering, to some extent, can overcome the limitations of genetic exchange in conventional breeding. Consequently, it considered as an effective method for improving modern crop breeding for B. napus. Since crop stress resistance is a polygenic complex trait, only by multi-gene synergistic effects can effectively achieve the comprehensive stress resistance of crops. Hence, in the present study, five stress resistance genes, NCED3, ABAR, CBF3, LOS5, and ICE1 were transferred into B. napus. Compared with wildtype (WT) plants, the multi-gene transformants K15 exhibited pronounced growth advantage under both normal growth and stress conditions. Additionally, K15 plants also showed significantly higher resistance response to multiple stresses at seed germination and seedling stages than WT plants. Furthermore, K15 plants had significantly higher leaf temperature and significantly lower stomatal aperture and water loss rate than WT plants, which indicated that the water-holding capacity of K15 plants was significantly superior to that of WT plants after stress treatment. In addition, K15 plants had significantly higher abscisic acid (ABA) content and significantly lower malondialdehyde (MDA) content than WT plants. In conclusion, the above results suggested that multi-gene co-expression could rapidly trigger plant stress resistance, reduce the stress injury on plants and synergistically improve the comprehensive resistance of B. napus.