In this paper, the interaction of lomefloxacin (LMF) and/or cefazolin (CFZ) with bovine serum albumin (BSA) was studied by fluorescence quenching in combination with UV-vis spectroscopic method under simulative physiological conditions. The fluorescence quenching constants, binding distance, and binding constants for BSA–LMX and/or CFZ systems were determined. The fluorescence quenching of BSA by addition of LMF and/or CFZ is due to static quenching and energy transfer. The ratio of binding constants (Ka) for BSA–LMF to BSA–CFZ equals to 22.41. In the presence of CFZ (LMF), the binding distance of BSA–LMF (BSA–CFZ) decreased from 4.44 to 2.45 nm (1.94 to 1.48 nm), and the binding constant (Ka) of BSA–LMF (BSA–CFZ) increased from 1.21×104 to 2.72×104 L mol–1 (5.40×102 to 1.20×103 L mol–1). Two-coexisting CFZ and LMF may lead to the need for more doses to achieve therapeutic effect. Circular dichroism spectra, synchronous fluorescence, and three-dimensional fluorescence studies showed that the presence of LMF or CFZ could change the conformation of BSA during the binding process, and the presence of coexisting LMF and CFZ could change the conformation of BSA further, here LMF was reigning.