L-Ergothioneine (L-EGT) possesses excellent antioxidant activity and has been used in the food, pharmaceuticals and cosmetics industries. In this study, a new efficient and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established for the quantitative determination of L-EGT in food. The sample was extracted with methanol-water (70:30, V/V), separated by hydrophilic interaction liquid chromatography (HILIC) and detected by triple-quadrupole mass spectrometry. Validation studies were carried out on different product and the limit of quantitation was 20 μg/kg (milk, alcohol-free beverages, dairy products) and 40 µg/kg (cereal bars, chocolate). Excellent linearity (correlation coefficient (R2) ≥ 0.999) was achieved for L-EGT quantification in the range of 5–200 ng/mL. The recoveries of the method (83.7%−107.5%) and the relative standard deviation (RSD, 0.88%−6.84% (n = 6)) meet the performance criteria required for the determination of L-EGT in food. Finally, the applicability of the method was tested by analysing actual samples. In general, the method developed is simple, reliable, accurate, and stable and could be useful for routine analyses of L-EGT in food.