Abstract Microfluidics devices for co-culturing neurons and oligodendrocytes represent an important in vitro research tool to decipher myelination mechanisms in health and disease and in the identification of novel treatments for myelin diseases. In reported devices using primary rodent cells, the spontaneous formation of myelin sheaths has been challenging and random orientation of neurites impede the analysis of myelination. Furthermore, fabrication methods for devices show limitations, highlighting the need for novel in vitro cell-based myelination models. In the present study, we describe a compartmentalized cell culture device targeted for neuron-oligodendrocyte co-culturing and myelination studies. In the device, neurites from primary rat dorsal root ganglion (DRG) neurons were capable of forming aligned dense networks in a specific compartment that was physically isolated from neuronal somas. Co-culture of rat DRG neurons and oligodendrocytes, a well-known model to study myelination in vitro, led to interactions between oligodendrocytes and neurites in the device, and the deposition of myelin segments in an aligned distribution was spontaneously formed. For the fabrication of the device, we present a new method that produces polydimethylsiloxane (PDMS)—based devices possessing an open compartment design. The proposed fabrication method takes advantage of an SU-8 photolithography process and 3D printing for mould fabrication. Both the microscale and macroscale features are replicated from the same mould, allowing devices to be produced with high precision and repeatability. The proposed device is applicable for long-term cell culturing, live-cell imaging, and by enhancing aligned myelin distribution, it is a promising tool for experimental setups that address diverse biological questions in the field of myelin research.