To date, the bacterial DNA topoisomerases are one of the major target biomolecules for the discovery of new antibacterial drugs. DNA topoisomerase regulates the topological state of DNA, which is very important for replication, transcription and recombination. The relaxation of negatively supercoiled DNA is catalyzed by bacterial DNA topoisomerase I (topoI) and this reaction requires Mg²⁺. In this report, we first quantitatively studied the intermolecular interactions between Escherichia coli topoisomerase I (EctopoI) and pBAD/Thio supercoiled plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (K_d) for EctopoI–pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg²⁺ on the catalysis of EctopoI–pBAD/Thio reaction. A slightly higher equilibrium dissociation constant (∼15 nM) was obtained for Mg²⁺ coordinated EctopoI (Mg²⁺EctopoI)–pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (k_d) for Mg²⁺EctopoI–pBAD/Thio interactions (∼0.043 s⁻¹), compared to EctopoI–pBAD/Thio interactions (∼0.017 s⁻¹). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg²⁺ and furthers the understanding of importance of the Mg²⁺ ion for bacterial topoisomerase I catalytic activity.