A fluorogenic probe for the specific detection of IMP-1 β-lactamase activity has been developed. This imaging reagent features a unique trans-acetylamino cephalosporin as an enzymatic recognition moiety, exhibiting excellent selectivity to IMP-1 β-lactamase over other β-lactamases, including serine- and metallo-β-lactamases. The selective activation of the probe by IMP-1 β-lactamase leads to over 30-fold enhancement in the fluorescence intensity, which allows enzyme activity to be reported with high sensitivity.