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.　©　2018　American　Chemical　Society.