Phosphorescent 4-morpholineethanesulfonic acid-capped Mn-doped ZnS (MES-MnZnS) quantum dots were developed for sensitively and selectively detecting L-tyrosine (L-Tyr) in human urine. L-Tyr can effectively quench the phosphorescence of MES-MnZnS even at low concentration levels. The mechanism of phosphorescence quenching is ascribed to electron transfer from MES-MnZnS QDs to L-Tyr. Under optimal conditions, the phosphorescent probe allowed a highly sensitive detection of L-Tyr with a wide linear range of 0.5–100 μM and a low detection limit of 0.22 μM. The developed method offers good selectivity over other compounds and was applied to determine L-Tyr in real urine samples with quantitative spike recoveries from 96.7% to 106.2%.