Polystyrene (PS) is one of the most commonly used thermoplastic materials worldwide and plays a ubiquitous role in today’s biomedical and life science industry and research. The main advantage of PS lies in its facile processability, its excellent optical and mechanical properties, as well as its biocompatibility. However, PS is only rarely used in microfluidic prototyping, since the structuring of PS is mainly performed using industrial-scale replication processes. So far, microfluidic chips in PS have not been accessible to rapid prototyping via 3D printing. In this work, we present, for the first time, 3D printing of transparent PS using fused deposition modeling (FDM). We present FDM printing of transparent PS microfluidic channels with dimensions as small as 300 µm and a high transparency in the region of interest. Furthermore, we demonstrate the fabrication of functional chips such as Tesla-mixer and mixer cascades. Cell culture experiments showed a high cell viability during seven days of culturing, as well as enabling cell adhesion and proliferation. With the aid of this new PS prototyping method, the development of future biomedical microfluidic chips will be significantly accelerated, as it enables using PS from the early academic prototyping all the way to industrial-scale mass replication.