In recent decades, the rise of β-lactamases has substantially led to the emergence and wide spread of antibiotic resistance posing a serious global health threat. There is growing need for the development of rapid, cost-effective and user-friendly diagnostic assays for the accurate detection of β-lactamases to optimize patient outcomes and prevent the spread of multidrug-resistances. In this article, we present a poly-dimethylacrylamide (PDMA)-based surface functionalization to immobilize β-lactam antibiotics and β-lactamase inhibitors of different subclasses. Immobilization was induced via UV-crosslinking through C,H-insertion reactions. The functional coatings were successfully applied in a highly efficient assay for the determination of recombinant β-lactamases as well as β-lactamases isolated from clinically relevant bacterial strains. Thus, this method describes an innovative approach with several significant benefits for diagnostic applications: the creation of specific detection platforms tailored for β-lactamase activity, the development of high-throughput diagnostic assays and benefits regarding stability and shelf-life. Furthermore, this method is highly adaptable to other surfaces, antibiotics, and analytes, offering far-reaching implications for various biomedical, environmental, and antimicrobial applications.