Soybean (Glycine max (L.) Merr.) is one of the most important crops with economic value as a source of protein and vegetable oil for human food and animal feed. In recent years, rapidly developed genome editing techniques have shown widespread application prospects for gene function studies and for improving important agronomic traits in many crops. Therefore, it is important to establish a highly efficient method for protoplast isolation and transient expression systems in soybeans. In this study, we established an efficient method for protoplast isolation and its application to transient gene expression in Korean soybean cultivars. The protoplasts were isolated from leaves, epicotyls, hypocotyls, cotyledons, and etiolated hypocotyls using various combinations of enzyme mixtures. We found that high-quality and large amounts of protoplasts were isolated from the etiolated hypocotyls when incubated for 8 h under conditions of 0.5% cellulase, 0.5% pectinase, and 1% viscozyme. In addition, we observed a high transfection efficiency of green fluorescent protein using etiolated hypocotyl protoplasts. Taken together, our protoplast isolation and transfection method is highly efficient and can be used for gene function and molecular analysis to better understand the biological and physiological processes in soybean.