Metallo‐β‐lactamases (MβLs) hydrolyze and inactivate β‐lactam antibiotics, are a pivotal mechanism conferring resistance against bacterial infections. SMB‐1, a novel B3 subclass of MβLs from Serratia marcescens could deactivate almost all β‐lactam antibiotics including ampicillin (AMP), which has posed a serious threat to public health. To illuminate the mechanism of recognition and interaction between SMB‐1 and AMP, various fluorescence spectroscopy techniques and molecular dynamics simulation were employed. The results of quenching spectroscopy unraveled that AMP could make SMB‐1 fluorescence quenching that mechanism was the static quenching; the synchronous and three‐dimensional fluorescence spectra validated that the microenvironment and conformation of SMB‐1 were altered after interaction with AMP. The molecular dynamics results demonstrated that the whole AMP enters the binding pocket of SMB‐1, even though with a relatively bulky R1 side chain. Loop1 and loop2 in SMB‐1 undergo significant fluctuations, and α2 (71–73) and local α5 (186–188) were turned into random coils, promoting zinc ion exposure consistent with circular dichroism spectroscopy results. The binding between them was driven by a combination of enthalpy and entropy changes, which was dominated by electrostatic force in agreement with the fluorescence observations. The present study brings structural insights and solid foundations for the design of new substrates for β‐lactamases and the development of effective antibiotics that are resistant to superbugs.