Nanoimprint lithography (NIL) is suitable for achieving high uniformity and mass production. However, in conventional NIL, a stamp suitable for the substrate size is required to increase the substrate size. To address this issue, we fabricated nanostructures on a large-area substrate using step-and-repeat NIL after making a small stamp. A stamp was produced using glass, and a nano-pillar pattern with a diameter of 600 nm, an interval of 400 nm, and a height of 270 nm was used during the experiment. The area of the pattern on the stamp was 10 mm × 10 mm, and the step-and-repeat process was performed 25 times to transfer the nanostructures to a 4-inch substrate. In addition, stitch gaps were created between the patterns, which could decrease the performance upon future application. To minimize this stitch gap, a high-precision glass scale was attached to the stamp feeder to precisely control the position and to minimize the step difference. Moreover, an experiment was conducted to minimize the stitch gap by adjusting the movement interval of the stamp, and the stitch spacing was minimized by moving the stamp position by 9.97 mm. This approach will facilitate the manufacturing of large-area substrates and other structures in the future.