Understanding the intricate interplay between proteins and ligands is pivotal for rational drug development. This study investigates the binding of Ofloxacin to Hb using diverse biophysical techniques: fluorescence spectroscopy, UV-visible spectroscopy, molecular docking, molecular dynamics simulations, and electrochemical analysis. Spectrophotometric data confirm a static quenching mechanism in ofloxacin-Hb binding. Notably, ofloxacin-Hb binding exhibits a significant change in free energy (−17.134 kJ/mol) and strong affinity via molecular docking was also observed (−8.2 kcal/mol). Molecular dynamics simulations validated these results, showing a change in free energy of −91.093 kJ/mol with minimal error of 13.508 kJ/mol, supporting a plausible interaction. Additionally, cyclic voltammetry elucidated dynamic interactions between Hb and ofloxacin. This comprehensive approach provides insights into drug-protein binding dynamics, crucial for refining drug development strategies.