A new approach of chemically immobilizing antibody within a pattern based on thin-film cracking is presented. An adjustable pattern width is achieved with resolutions varied from nano- to microscale by using loading stress on thin-film coated elastomer substrate in both one and two dimensions. By introduction of solution or chemical vapor deposition approaches, antibodies were covalently immobilized in the channels. To demonstrate the bioactivity, specificity, and response rate of antibody patterned structure, scanning electron microscopy was used to enumerating bacteria. The chemically coupled antibody is found to retain its specificity when incubated with different bacteria solutions. Trichloro(1H,1H,2H,2H-perfluoroctyl)silane coating on nonsensing regions exhibits a distinguished bacteria-resistant function that is beneficial for providing a low intrinsic background signal in detection. This technique shows a great potential for applications in the fields of sensing and tissue engineering.