Ses i 3 is one of the major allergens in sesame, and this study aimed to investigate the effect and mechanism of ultrasound-assisted glycation on the potential allergenicity of Ses i 3. Results showed that the IgG and IgE binding of Ses i 3 was significantly reduced after ultrasound-assisted glycation, and the KU812 cell degranulation results showed a significantly decrease of degranulation in the ultrasound-assisted glycation group, suggesting a reduction in the allergenicity of Ses i 3. Subsequently, results from the contents of free amino groups and available lysine showed that ultrasound pretreatment facilitated the glycation reaction. Moreover, five linear epitopes were predicted, with S-5 (AA 571-582) being identified as the effective B cell linear epitope through solid-phase peptide synthesis and serological experiments. Its spatial location was also determined using a AlphaFold 3 model. Besides, inhibited competitive ELISA results indicated that the addition of inhibitor (S-5) didn't reduce the IgE binding of Ses i 3 in the ultrasound-assisted glycation group, suggesting that ultrasound-assisted glycation might have masked or destroyed the S-5 epitope. Ultraviolet (UV) spectra and intrinsic fluorescence emission spectrum results revealed a blue shift in the maximum absorption peaks, along with a significant reduction in surface hydrophobicity, indicating substantial changes in both secondary and tertiary structures. In conclusion, this study demonstrated that ultrasound-assisted glycation effectively reduced the allergenicity of Ses i 3, with the reduction being closely linked to changes in the S-5 epitope and structures. These findings will provide a basis for the development of hypoallergenic sesame products.