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This paper describes the development of a microfluidic device for axon rientation. The device was fabricated with poly (dimethylsiloxane) by soft lithography and replica molding. There were two separated rooms in the device, and a control system and stable concentration gradient system were maintained in each room for at least 24 h. Finally, to demonstrate a practical application of the device, neuronal cells were cultured in both cell rooms, and he growth of cells and formation of axons were monitored under a concentration gradient of cytochalasin D for 8 h. Quantitative analysis of the rientation of axons was analyzed, and the data were plotted in a polar graph as a function of the direction. A figure of 33.8% of positive responding neurons were found in the cytochalasin D gradient room, while that figure was 23.3% of cells in control room. The extent of axon orientation shows a considerable difference between the two rooms, and the device developedhere is capable of investigating axon orientation and can be used for other biological applications.