To assess the toxicity of residual marbofloxacin from animal‐derived food, the interaction characteristics of marbofloxacin to bovine/human serum albumins (BSA/HSA) were explored using spectroscopic methods combined with molecular modelling. According to fluorescence spectra and time‐resolved fluorescence spectra measurements, quenching of BSA/HSA fluorescence induced by marbofloxacin was characterized as static quenching. A 1:1 ground‐state complex of marbofloxacin to BSA/HSA was formed with binding constant (Ka) 1.66 × 104/9.74 × 103 M−1 at 291 K. The location of marbofloxacin binding at site I within BSA/HSA was clarified by site marker competitive experiments. Molecular modelling demonstrated that the binding region for marbofloxacin to BSA and HSA were at site I with the lowest binding free energies of −22.86 and −21.60 kJ mol−1, respectively. Hydrogen bonds and van der Waals forces were dominantly involved in the spontaneous binding. Nonradiation energy transferred from BSA and HSA to marbofloxacin, due to the close distance (r0) between marbofloxacin and Trp residues of BSA (4.28 nm) and HSA (3.34 nm). As explained by circular dichroism (CD) spectra, an increased BSA/HSA α‐helix structure was observed after binding to marbofloxacin. Ultraviolet–visible (UV–vis) and Fourier transform infrared (FT‐IR) spectra suggested that conformation of the two proteins was altered by marbofloxacin.