The selection of a quality marker is crucial for quality control of Lycium barbarum L. fruits, which are widely used in the food and drug industries, especially as herbal medicine, and functional food. However, using betaine, the quality marker in the current standard, for discrimination of L. barbarum fruits lacks specificity. In this study, a vitamin C derivative, 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), was first used as an antioxidant biomarker instead of betaine for the quality control of L. barbarum fruits from different regions. Three-factor-three-level response surface methodology was used to optimize extraction conditions, which indicated that the optimal conditions of AA-2βG were 75% ethanol as extraction solvent, 1:25 of solvent-to-solid ratio, and 30 min for extraction time. Hydrophilic interaction liquid chromatography method was applied for the quantification of AA-2βG. The results showed that the content of AA-2βG in L. barbarum fruits from different regions was above 9 mg/g except for samples of NLB 8, NLB 30 and NLB 74. All samples were classified into three clusters with Hierarchical Cluster Analysis, and most of them belong to cluster Ⅲ with the content of AA-2βG in the range of 9.04–11.8 mg/g.