For the multiple and rapid detection of β-lactams, the broadly specific penicillin-binding protein (PBP) that recognizes the β-lactam structure was prepared. A chromatographic strip based on europium chelate-loaded fluorescent microspheres was assembled with a goat anti-mouse antibody (C line) and penicillin coating (T line). The penicillin coating competes with free β-lactam antibiotics to bind PBP-labeled fluorescent microspheres. The strip can theoretically detect all kinds of β-lactams, including amoxicillin, ampicillin, oxacillin, ceftazidime, lenampicillin, cefoperazone, sultamicillin, cefotaxime, ceftriaxone, sulbenicillin, piperacillin, cephalothin, flucloxacillin, and mezlocillin, in samples within 10 min. The europium chelate-labeled lateral flow assay does not cross-react with other antibiotics, including chloramphenicol, tetracycline, sulfamethazine, enrofloxacin, gentamicin or lincomycin. In short, we developed a very useful method for preliminary screening of β-lactams.