This work describes a large-scale preparation method of a honeycomb microstructure surface (HMS) based on siliconeacrylate copolymers (PSiA) with abundant SiOH and organic amine. The mean diameter and depth of the honeycomb microstructures are controllable via changing the species and concentration of the organic amine as well as the siliconeacrylate mass ratio of copolymer. The formation mechanism of HMS was investigated, and the results showed that the tiny liquid droplets of organic amine acted as liquid state cores for the formation of honeycomb microstructures. The zoospore settlement and diatom attachment assay demonstrated that the cell density of organisms is lower on the microstructure surface than that on the smooth surface, confirming its preferable antifouling properties. Therefore, this work demonstrates a feasible approach of preparing HMS with controllable pore sizes via changing its composition, which can be used as good candidates for environmental-friendly antifouling surfaces.