Abstract: Based on the sensitization effect of bovine serum albumin (BSA) on doxycycline (DC), a simple, highly sensitive and selective turn-on fluorescence assay is developed for the detection of DC content. Due to the sensitization effect, the fluorescence signal of DC significantly enhances in the presence of BSA. It might be ascribe to that DC molecules could enter the hydrophobic cavity of BSA, and the DC rotation is restricted, leading to the fluorescence enhancement of DC. Under the optimized experimental conditions, the linear range of DC detection is 20 nmol/L~40 μmol/L, and the limit of detection is 5 nmol/L (R_SN=3). In addition, this method has been applied for the determination of DC in milk samples.