Understanding the functional contributions of myelin maturation is essential in the treatment of peripheral nerve diseases. However, little is known regarding sequential changes in the action-potential conduction velocity (CV) during the myelin maturation. This lack of knowledge is primarily because of the difficulty in evaluating the CV in individual axons included in the bundle. In this study, we developed a neuron/Schwann cell (SC) coculture device for evaluating the myelin-regulated changes in the CV by combining a microtunnel-electrode technique with a surface patterning method. A stripe pattern of matrigel for guiding axon growth was aligned on an electrode set covered by the microtunnel. Immunocytochemical staining demonstrated that more than 95% of the cells attached onto the stripe pattern and P0-positive myelin sheaths were formed in the microtunnel-covered area. Electrical stimulation evoked activities in the axons and the activities were separately recorded, indicating that our device provides the CV in individual axons. Furthermore, activity with the CV two times higher (1.02 m/s) than that of the unmyelinated axons appeared after the start of myelin formation, suggesting that the activity of partially myelinated axons can be detected. These results indicate that our approach is effective for investigating sequential changes in the CV with myelin maturation.