Class C β-lactamases mediate antibiotic resistance in bacteria by efficiently hydrolyzing a broad range of β-lactam antibiotics. With their clinical significance and the lack of commercially available effective inhibitors, development of class C β-lactamase inhibitors has become one of the recent hot issues in the pharmaceutical industry. In this paper, we report the protein engineering of a fluorescent Amp C β-lactamase mutant designated as V211Cf for the in vitro screening of class C β-lactamase inhibitors. When a fluorescein (f) was incorporated at the entrance of the enzyme's active site (position 211), Amp C β-lactamase from Enterobacter cloacae P99 was tailor-made into a novel fluorescent biosensing protein that could display a fluorescence enhancement upon binding with its β-lactam substrates/inhibitors. With its catalytic activity close to the wild-type level, V211Cf can act as a "natural" fluorescent drug target for screening small binding molecules. In addition, V211Cf can allow specific detection for its active-site binding molecules and discriminate them from nondruglike molecules in the screen. Furthermore, V211Cf is amenable to a high throughput format. Taken together, V211Cf demonstrates the potential as an efficient tool for screening class C β-lactamase inhibitors and facilitates the discovery of therapeutics that can combat the clinically important class C β-lactamases.