Allergen Glb33 is an important allergen in rice that can cause allergic reactions such as asthma and atopic dermatitis. However， knowledge of the content in rice is sparse. In the present work， an absolute protein quantification method was established for allergen Glb33 in rice samples using liquid chromatography-tandem mass spectrometry. After extraction of allergen Glb33 from rice grains using salt solution， the isotope-labeled peptide internal standard was added to the extract， followed by enzymatic digestion with trypsin. The signature peptide and its isotope-labeled analogue from the tryptic hydrolysates of allergen Glb33 and the internal standard were detected by liquid chromatography-tandem mass spectrometry. The quantitative bias caused by tryptic efficiency and matrix effect was corrected by using two isotope-labeled standard peptides. The method exhibited good linearity in the range of 1-200 nM， with coefficients of determination of R > 0.998. A high sensitivity was observed， with a limit of quantification of 0.97 nM. Mean recoveries obtained from different rice matrices ranged from 82.7%-98.1% with precision <8.5% in intraday trials ( n = 6)， while mean recoveries were from 75.1%-107.4% with precision <14.6% in interday trials ( n = 14). The developed method was successfully applied to the analysis of allergen Glb33 in 24 different rice cultivars.