Recording axonal conduction will be a strong tool to continuously evaluate Na channel expression of cultured neurons. However, little is known about the relationship between ion channel expression and axonal conduction velocity. In this study, we aim to develop a method to evaluate the relationship. A microdevice was developed with photo- and soft-lithography. Cortical neurons were cultured, and activity propagating along axons was recorded. After spike sorting, mixed signal from multiple axons were sorted into clusters of individual axons. Axons were treated with non-selective and subtype specific Na channel blockers, and changes in conduction delay were evaluated. TTX and lidocaine increased conduction delay with a different manner, suggesting that the different affinity and binding kinetics can be detected with the device. Moreover, although Nav 1.2 blocker increased the conduction delay and eventually clocked the conduction at around IC50, the other blockers did not. This result suggests that Nav 1.2 is dominant for the conduction along unmyelinated cortical axons. Overall, our device should be a feasible tool for elucidating Na channel properties by axon-targeted recording.