We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening.