Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming. In order to establish a universal protoplast-based transient transfection system for verifying activities of genome editing vectors containing targets in Brassica, we systematically optimized factors affecting protoplast isolation and transient gene expression. We established an efficient protoplast-based transient gene expression system (PTGE) in Chinese cabbage, achieving high protoplast yield of 4.9 × 105 · g−1 FW, viability over 95%, and transfection efficiency of 76%. We showed for the first time that pretreatment of protoplasts with a hypotonic MMG could significantly enhance the transfection efficiency. Furthermore, protoplasts incubated at 37 °C for 6 min improved the transfection efficiency to 86%. We also demonstrated that PTGE worked well (more than 50% transfection efficiency) in multiple Brassica species including cabbage, Pak Choi, Chinese kale, and turnip. Finally, PTGE was used for validating the activities of CRISPR/Cas9 vectors containing targets in Chinese cabbage, cabbage, and pak choi, demonstrating the broad applicability of the established PTGE for genome editing in Brassica crops.