There have been considerable efforts to engineer three-dimensional (3D) microfluidic environments to enhance cellular function over conventional two-dimensional (2D) cultures in microfluidic chips, but few involve topographical features, such as micro/nano-grooves, which are beneficial for cell types of cardiac, skeletal and neuronal lineages. Here we have developed a cost-effective and scalable method to incorporate micro-topographical cues into microfluidic chips to induce cell alignment. Using commercially available optical media as molds for replica molding, we produced large surface areas of polydimethylsiloxane (PDMS) micro-grooved substrates and plasma-bonded them to multiple microfluidic chips. Besides aligning a 2D monolayer of cells, the micro-grooved substrate can align 3D cellular constructs on chip. C2C12 mouse myoblasts were cultured three-dimensionally in a microfluidic chip with incorporated PDMS micro-grooved substrate remodeled into an aligned 3D cellular construct, where the actin cytoskeleton and nuclei were preferentially oriented along the micro-grooves. Cells within the 3D cellular constructs can align without being in direct contact with the micro-grooves due to synergism between topography and fluid shear stress. Aligned C2C12 3D cellular constructs showed enhanced differentiation into skeletal muscles as compared to randomly aligned ones. This novel method enables the routine inclusion of micro-topographical cues into 2D or 3D microfluidic cultures to generate relevant physiological models for studying tissue morphogenesis and drug screening applications.