In this paper, the mechanism of the binding interaction between residual ciprofloxacin (CIP) and dissolved organic matter humic acid (HA) in natural water environment was systematically investigated. Various spectral analysis technologies, including the three-dimensional excitation-emission matrix spectra, fourier transform infrared (FTIR) spectra, two-dimensional correlation spectra (2D-COS) and liquid nuclear magnetic resonance (¹H NMR) spectra techniques, were adopted to reveal the mechanism of the binding interaction between CIP and HA. The results suggested that the binding interaction between CIP and HA resulted in significant fluorescence quenching, and the binding interaction tended to balance after 6 h monitored by ultraviolet (UV) differential spectroscopy. Based on the FTIR analysis, the hydroxyl, carboxyl and ketone groups of HA were the main contributors to the binding interaction, and the results of 2D-COS indicated that the ketone groups preferentially participated in binding interaction compared to carboxyl groups. The H protons on the piperazine ring of CIP were identified as the main binding sites by the ¹H NMR, and the comparison of fluorescence quenching rate of other fluoroquinolone antibiotics (norfloxacin and levofloxacin) with similar structures also verified that the piperazine ring played a crucial role in binding interaction. Additionally, the influence of ionic strength (IS, range 0.001—0.1) on the binding interaction was ignorable compared with pH. The fluorescence quenching rates increased firstly and then decreased with the increase of pH (range 3—11), and the fluorescence quenching rate was up to 70% at pH 5. Moreover, the fluorescence quenching rates were positively correlated with the valence state of metal ions (order of influence followed: Al³⁺>Zn²⁺, Co²⁺, NH₄⁺, Ca²⁺>Mn²⁺, Na⁺, K⁺). This study provides the theoretical basis and effective detection technologies for monitoring the migration and transformation of residual antibiotics in the natural water environment.