Protein glycation reaction based on traditional heating methods has disadvantages, such as long reaction time consumption and easy to produce harmful advanced glycation end products. In this study, the ovomucoid (OVM)-reducing sugar (1∶0.03, W/W) system served as the research object, effects of superheated steam, a new high-temperature treatment technology, on the OVM glycation and protein structure were studied. The results showed that the glycation of OVM was induced by a short time (1~3 min) of superheated steam treatment (110 and 120 ℃), and the sequence of glycation reaction activity of different sugars was ribose (pentose) > glucose (hexose) > lactose (disaccharide). After glycation, the free amino content of protein decreased from 19.97 mg·mL-1 to the minimum of 2.93, 5.04 and 6.69 mg·mL-1, respectively. The ultraviolet spectra of OVM glycation products under different processing conditions showed no significant changes in wave displacement. However, the maximum absorption had some changes, with most of them were reduced, indicating OVM globulin molecular structure was altered, and the chromophore groups (phenylalanine and tyrosine) of OVM were masked by the reducing sugars. The effect was most significant under ribose glycation. After glycation, the fluorescence intensities were significantly decreased, in which the ribose glycation decreased the most, followed by glucose and lactose, indicating that the fluorescence quenching occurred, which resulted from the folding of exposed fluorophore in protein. In addition, reducing sugar as a quenching agent penetrated the protein framework and reacted with fluorophore, inhibiting the fluorescence emission. Fourier transform infrared spectroscopy showed that, after superheated steam treatment, the protein peak at 3 300 cm-1 became narrow, indicating that the content of N-H decreased or a reaction occurred, thus reducing the stretching vibration frequency of N-H. Some fingerprint peaks in the vicinity of 2 000～2 500 and 500 cm-1appeared, indicating that the protein degradation or glycation middle product formation occurred. MALDI TOF MS analysis showed that there were about 6 glycation reaction sites in each the OVM-ribose system and OVM-glucose system, and about 2 glycation reaction sites in the OVM-lactose system. Moreover, after glycation, OVM dimer, trimer and multimer were formed. Ribose and glucose produced the most significant polymers with OVM, while lactose produced fewer polymers with OVM. This study can provide technical and theoretical guidance for the glycation reaction featured by trace amounts of sugar participation, short time consumption, and application of superheated steam in food processing.