The pollution caused by the abuse of antibiotics has posed a serious threat to the ecological environment and human health, so development of effective strategies for degradation and disposal of antibiotic residues is urgently needed. In this work, penicillinase, a kind of β-lactamase, was immobilized into zeolitic imidazolate framework-8 (ZIF-8) by self-assembly method and the catalytic performance of the β-lactamase@ZIF-8 porous materials for degradation of penicillins has been investigated by high performance liquid chromatography coupled with mass spectrometry. The results illustrated that the catalytic activity of the encapsulated enzyme was significantly enhanced comparing with that of free enzyme. Meanwhile, the β-lactamase@ZIF-8 exhibited excellent stability under denaturing conditions including high temperature, organic solvent and the enzyme inhibitor. The catalytic degradation mechanism of the β-lactamase@ZIF-8 for penicillins has been probed and verified, and it has been found that the Zn (II) ion on ZIF-8 frameworks could form the complex with the target molecule, which weakened the bond of the four-membered β-lactam ring in the penicillin molecule, and thus enhanced the degradation efficiency of the enzyme. This work provided a promising strategy for eliminating the penicillin residues in water environment. Display omitted •The β-lactamase has been encapsulated into ZIF-8 MOFs by a self-assembly method.•The β-lactamase@ZIF-8 MOFs possessed excellent stability under denaturing conditions.•Catalytic activity of enzyme encapsulated into ZIF-8 MOFs was significantly enhanced.•The catalytic degradation pathway of penicillins was proposed and verified.