In this paper, we describe a microfluidic platform that enables three dimensional cell culture within defined microenvironments towards a study of axon-glia interaction. The microdevice can offer both 3D axon isolation from cell body and 3D spatial cell separation between neuron and astrocyte. It consists of large fluidic channels for media supply and small channels for collagen filling as a three dimensional ECM (extracellular matrix). Astrocytes were seeded in collagen gel through small fluidic channels and neuron cells were aggregated at an openings of small channel to large channels. This method separated neuronal soma from axon as well as segregatedneuron body from astrocyte. The preliminary study on the effect of astrocyte to the axon growth showed that axon growth was faster and richer in co-culture condition without growth factor. This 3D co-culture chip can provide a new method to observe the effect of astrocyte on the axon growth in three dimensional culture system.