Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and a lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transient transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. Unfortunately, there is no standardized method for Chinese cabbage. Hence, in this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were optimally isolated following a 4-h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) Macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl₂, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with 40 μg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study will be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.