To evaluate the effect of glucose and fructose on the glycation of β-lactoglobulin (β-Lg) in intermediate-moisture food (IMF), model systems consisting of β-Lg, glucose/fructose/sorbitol, glycerol, and water were established. All systems were stored at 25 and 35 °C for 2 months. The progress of the Maillard reaction and the mass change of β-Lg were investigated by the browning assay and gel electrophoresis, respectively. Meanwhile, liquid chromatography-mass spectrometry (LC-MS) and data-independent acquisition LC-MS (LC-MS(E)) were used to monitor the glycation extent and the glycated sites of β-Lg. The results indicated that glucose had a higher reaction activity of glycation than fructose, but both sugars had similar preference on the glycation site for β-Lg. The ranking order from high to low for the 9 detected glycated sites was L 1, K 91 > K 47 > K 70, K 77, K 83, K 100 > K 75 > K 135 for both sugars.