Polyurethanes (PU) are a broad class of polymers that offer good solvent compatibility and a wide range of properties that can be used to generate microfluidic layers. Here, we report the first characterization of a commercially available Shore 80D polyurethane (Ultraclear™ 480N) for biomicrofluidic applications. Studies included comparing optical clarity with Polydimethylsiloxane (PDMS) and using high-fidelity replica molding to produce solid PU structures from the millimeter to nanometer scales. Additionally, we report the first use of NanoAccel™ treatment in Accelerated Neutral Atom Beam (ANAB) mode to permanently roughen the surface of PU and improve the adhesion of breast cancer cells (MDA-MB-231) on PU. Surface energy measurements using Owens-Wendt equations indicate an increase in polar and total surface energy due to ANAB treatment. Fourier-transform infrared (FTIR) spectroscopy in attenuated total reflectance (ATR) mode was used to demonstrate that the treatment does not introduce any new types of functional groups on the surface of Ultraclear™ PU. Finally, applicability in rapid prototyping for biomicrofluidics was demonstrated by utilizing a 3D-printing-based replica molding strategy to create PU microfluidic layers. These layers were sealed to polystyrene (PS) bases to produce PU-PS microfluidic chips. Ultraclear™ PU can serve as a clear and castable alternative to PDMS in biomicrofluidic studies.