Functional microdomains consisting of multiple molecules have widespread applications. However, most of available methods reported so far have a common limitation for widespread practical use. Herein, we reported a facile method based on material-independent polydopamine surface chemistry to realize the area-selective immobilization of dual amine-/thiol-terminal bioactive molecules assisted by photolithography. We transferred the photoresist pattern to the polydopamine coating surface, and specific molecules were respectively covalently immobilized in the microdomain. The results demonstrated that molecular anchoring is area-selective and quantitatively controllable. Thus, this versatile method provides a new insight into the creation of regionally chemical multicomponent surfaces and could build a potential platform for promising application in sensors, molecular biology, and genetic diagnosis.