Abstract: A novel, simple and sensitive fluorescenct method for determination of cysteine in water, milk, milk powder, cabbage, apple, pear and radish was established based on rosamine fluorescent probe. The probe was constructed by rosamine as the signal reporter and phenylalkynyl as the reaction site. Through Michael addition and intramolecular reaction, the probe could detect cysteine with high selectivity and sensitivity. Results indicated that under the optimized conditions of phosphoric acid buffer concentration of 10 mmol/L, pH9.5, methanol/water volume ratio of 1:9 and room temperature reaction for 35 min, the probe selectively recognized cysteine, and its relative fluorescence intensity increased with the increasing of cysteine concentration. Furthermore, the fluorescence signal of the probe had a good linear relationship with cysteine in the concentration range from 18.0 to 70.0 μmol/L (R²=0.992) with detection limit of 0.18 μmol/L. Furthermore, the method was used to determinate cysteine in water, milk, milk powder, cabbage, apple, pear and radish with good recoveries (96.4%~102.97%) and RSD (0.1%～2%). This suggested that the approach would have high selectivity, sensitivity and the feasibility of cysteine detection in practical sample, as well as a good application prospect.