In order to investigate the regulation effect of hydrogel micropatterns of different sizes on cell behaviors, large-area polygons and polygonal microstructures of different sizes were achieved by a femtosecond laser maskless optical projection lithography technique, in combination with a large-area splicing method. The optimum processing conditions and the wettability of the microstructure are studied in detail. The coculture experimental results of fibroblasts with patterned substrates showed that the spatial confinement in the microstructure can change the cell morphology and effectively regulate the cell growth behaviors. In particular, in the small polygonal and polygonal star microstructure, the nucleus tends to fall into the concave lacunae of the microstructure, and the spreading of cytoskeleton gradually tends to be consistent with the morphology of microstructures. This study indicates that the size of microstructural pattern units is very important for inducing cell behavior and function, which would provide a new technology and new method for cell research in vitro by using biocompatible hydrogel microstructures.