Metallo-β-lactamases (MβLs) could hydrolyze almost all β-lactam antibiotics, the primary mechanism resulting in drug resistance against bacterial infections. This has become a substantial concern due to the lack of clinically approved inhibitors. SMB-1 from Serratia marcescents is a novel B3 subclass MβL that inactivates almost all β-lactam-containing antibiotics. The interaction mechanism between carbapenem antibiotic imipenem (IMIP) and Metallo-β-lactamase SMB-1 was ascertained in this paper using endogenous fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy and molecular docking methods. The quenching spectrum results demonstrated that IMIP quenched endogenous fluorescence of SMB-1, and the quenching mechanism was a combination of dynamic and static quenching, of which static quenching is the core one; the binding constant Ka was 16.11×103 L·mol-1 (277 K), indicating a strong binding force between them; the thermodynamic parameters in the binding process obtained from the Van’t Hoff equation ΔG<0, ΔH=-79.65 kJ·mol-1, ΔS=-238.69 J·mol-1, illustrating that the binding was driven by both enthalpy and entropy changes and hydrogen bonding and van der Waals forces were the main forces; Moreover, the maximum emission wavelength of SMB-1 in synchronous fluorescence results was blue shifted by 4.4 and 2.9 nm with increasing IMIP concentration, revealing that Tyr and Trp residues were involved in both. The significant decrease of Peak B and Peak C intensity of SMB-1 with IMIP introduced in the three-dimensional fluorescence spectra indicated that the microenvironment and conformation of SMB-1 changed after the interaction with IMIP, which is consistent with the synchronous fluorescence results. Furthermore, the β-lactam ring of IMIP entered the binding pocket of SMB-1 in the molecular docking results, while the side chain was located outside the active pocket due to the spatial site block effect, inferring that SMB-1 mainly recognized the core structure of IMIP and interacted weakly with its R2 side chain; the amino acid residues involved in the interaction with IMIP including Ser175, Thr177, Gln157, His215 and Glu217, implying that these amino acid residues with the two zinc ions in the active site are key factors in the design of SMB-1 inhibitors with strong affinity; the binding free energy was also negative, suggesting that the binding of both was a spontaneous exothermic process, which is consistent with the fluorescence results. Therefore, the present study provides insights into the recognition and binding of SMB-1 to IMIP, which may help design new substrates for β-lactamases and develop new antibiotics with resistance to superbugs.