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We present a sensitive and rapid screening method for the determination of β‐lactamase activity of antibiotic‐resistant bacteria, by designing a pH‐sensitive fluorescent dye‐doped mesoporous silica nanoparticle encapsulated with penicillin G as a substrate. When penicillin G was hydrolysed by β‐lactamase and converted into penicilloic acid, the acidic environment resulted in fluorescence quenching of the dye. The dye‐doped mesoporous nanoparticles not only enhanced the β‐lactamase‐catalyzed reaction rate but also stablized the substrate, penicillin G, which degrades into penicilloic acid in a water solution without β‐lactamase. Twentyfive clinical bacterial samples were tested and the antibiotic resistant and susceptible strains were identified. The proposed method may detect the presence of β ‐lactamases of clinically relevant samples in less than 1 hour. Moreover, the detection limit of β‐lactamase activity was as low as 7.8×10−4 U/mL, which was determined within two hours.

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