Direct laser writing on biodegradable polymer to create microchannels for aligning cells is presented here. This technique offers the advantages of ease-of-manufacturing, ease-of-design, high-speed single-step fabrication, and noncontacting to the material. In this work, microchannels of 100 microm width, 100 microm depth, and 50 microm intervals were created on a biodegradable polymer film directly using a Ti-sapphire femtosecond pulsed laser. Multiscale topological features were achieved as a result of the laser beam-material interaction. These topological features were used to guide cell alignment in the microchannels. We present results on the morphology of poly(L-lactide-co-epsilon-caprolactone) copolymer micromachined by femtosecond laser and demonstrate the attachment and alignment of C2C12 myoblast cells in the microchannels. C2C12 cells exhibited favorable attachment in the channels after 1 day of seeding. High degree of alignment was observed after 4 days as cells proliferated into a confluent patch inside the channels. This work demonstrated the potential of wavy surface features combined with appropriate channel size for high-density cell alignment using direct laser writing. This method also offers the opportunity to incorporate multiscale topological guidance on other biodegradable polymer implants, such as vascular scaffolds and stents, which require directed cell organization.