Abstract In this study, the voltammetric behavior of the interaction between tilmicosin (TIM) and bovine serum albumin (BSA) was investigated using voltammetry and UV–Vis spectroscopy techniques. In the presence of BSA, the main reductive signal of TIM gradually decreased without the appearance of new peaks. The formation of a 1:1 complex between TIM and BSA were determined at different pHs (3, 7.4 and 11). This interaction was also supported by ultraviolet spectroscopy. After the addition of TIM into the BSA solution, at pH 3 and 7.4, the absorption band of BSA at 217 nm decreased, shifted to smaller wavelengths, and narrowed. However, at pH 11, the absorbance of BSA at 217 nm decreased without the shift of maximum absorption wavelength. The interaction between TIM and BSA was mainly sourced from electrostatic or hydrophobic (intercalation) interactions.