Organ-on-a-chip devices are gaining popularity in medical research due to the possibility of performing extremely complex living-body-resembling research in vitro. For this reason, there is a substantial drive in developing technologies capable of producing such structures in a simple and, at the same time, flexible manner. One of the primary challenges in producing organ-on-chip devices from a manufacturing standpoint is the prevalence of layer-by-layer bonding techniques, which result in limitations relating to the applicable materials and geometries and limited repeatability. In this work, we present an improved approach, using three dimensional (3D) laser lithography for the direct integration of a functional part—the membrane—into a closed-channel system. We show that it allows the freely choice of the geometry of the membrane and its integration into a complete organ-on-a-chip system. Considerations relating to sample preparation, the writing process, and the final preparation for operation are given. Overall, we consider that the broader application of 3D laser lithography in organ-on-a-chip fabrication is the next logical step in this field’s evolution.