Soybean is one of the most important crops because of its high protein and oil content. Previous studies have refined protoplast isolation methods for soybeans to enhance transfection efficiency. However, these methods have limitations due to the inconsistent number of viable protoplasts for various applications. In this study, we propose an optimized protoplast isolation method to overcome this challenge. Hypocotyls grown in the dark were selected to ensure rapid growth and a steady supply of plant materials. The hypocotyls were cut to 1–2 cm in length and halved longitudinally to achieve consistent protoplast yields. Our new hypocotyl cutting method demonstrated 1.5 times improved protoplast yield and improved protoplast viability compared to that of previous methods. The isolated protoplasts were purified using the sucrose density gradient purification method to remove residues while stacking viable protoplasts. Fluorescein diacetate (FDA) staining was performed to determine the proportion of healthy protoplasts throughout the process. Consequently, we propose a new protoplast isolation protocol that ensures a higher yield, better viability, and healthier conditions. This enhancement is expected to improve the efficiency of soybean transfection.