Biosensors have found applications in many sectors including the food industry, where cephalosporin detection has played an important role in reducing the incidence of cephalosporin contamination, ensuring food safety, and reducing the spread of antibiotic resistance. Taking advantage of the specific interaction between β-lactamase and its cephalosporin substrates/inhibitors, we previously constructed a biosensor based on a fluorescein-labeled class C β-lactamase mutant, V211Cf, for specific and reagentless detection of cephalosporins and class C β-lactamase inhibitors (Anal. Chem. 2011, 83, 1996-2004). Upon the addition of substrate/ inhibitor (i.e. the biosensor's analyte), the analyte induced a change in the local environment of the fluorescein molecule that was covalently tethered to a site close to the enzyme's active site (the 211 position), triggering a fluorescence enhancement of V211Cf. To improve the performance of V211Cf for better cephalosporin detection of the biosensor, we have developed Y150S/V211Cf, a derivative of V211Cf constructed by introducing the Y150S mutation to suppress the hydrolytic activity of V211Cf thereby improving the stability of the fluorescence signal. From our results, Y150S/V211Cf not only demonstrated improved fluorescence signal sustainability over V211Cf, but also showed a rapid response towards cephalothin (a first generation cephalosporin). These features make it feasible to of use Y150S/V211Cf for the rapid and specific detection of cephalosporins, and illustrate the possibilities for rational biosensor design of catalytically impaired fluorescent enzymes for rapid and sensitive analyte detection purposes.