The interaction of dexamethasone sodium phosphate (DEX-P) with bovine serum albumin (BSA) was studied by fluorescence quenching in combination with UV-Vis spectroscopic method under near physiological conditions. Fluorescence quenching rate constants and binding constants for BSA–DEX-P system were determined at different temperatures. The fluorescence quenching of BSA by DEX-P was due to static quenching and energy transfer. The results of thermodynamic parameters, ΔH (−161.0 kJ mol⁻¹), ΔS (−468.0 J mol⁻¹ K⁻¹) and ΔG (−21.54 to −16.86 kJ mol⁻¹), indicated that van der Waals interaction and hydrogen bonding played a major role in DEX-P–BSA association. Competitive experiments demonstrated that the primary binding site of DEX-P on BSA was located at site III in sub-domain III_A of BSA. The distance between BSA and DEX-P was estimated to be 1.23 nm based on the Förster resonance energy transfer theory. The binding constant (K_a) of BSA–DEX-P at 298 K was 2.239 × 10⁴ L mol⁻¹. Circular dichroism spectra, synchronous fluorescence and three-dimensional fluorescence studies showed that the presence of DEX-P could change the conformation of BSA during the binding process.