Emergence of antibiotic bacterial resistance has caused serious clinical issues worldwide due to increasingly difficult treatment. Development of a specific approach for selective visualization of resistant bacteria will be highly significant for clinical investigations to promote timely diagnosis and treatment of bacterial infections. In this article, we present an effective method that not only is able to selectively recognize drug resistant AmpC β-lactamases enzyme but, more importantly, is able to interact with bacterial cell wall components, resulting in a desired localization effect on the bacterial surface. A unique and specific enzyme-responsive cephalosporin probe (DFD-1) has been developed for the selective recognition of resistance bacteria AmpC β-lactamase, by employing fluorescence resonance energy transfer with an "off-on" bioimaging. To achieve the desired localization, a lipid-azide conjugate (LA-12) was utilized to facilitate its penetration into the bacterial surface, followed by copper-free click chemistry. This enables the probe DFD-1 to be anchored onto the cell surface. In the presence of AmpC enzymes, the cephalosporin β-lactam ring on DFD-1 will be hydrolyzed, leading to the quencher release, thus generating fluorescence for real-time resistant bacterial screening. More importantly, the bulky dibenzocyclooctyne group in DFD-1 allowed selective recognition toward the AmpC bacterial enzyme instead of its counterpart ( e.g., TEM-1 β-lactamase). Both live cell imaging and cell cytometry assays showed the great selectivity of DFD-1 to drug resistant bacterial pathogens containing the AmpC enzyme with significant fluorescence enhancement (∼67-fold). This probe presented promising capability to selectively localize and screen for AmpC resistance bacteria, providing great promise for clinical microbiological applications.