This study aims to evaluate the effect of ultrasonic pretreatment combined with glycation on the structural characteristics and antibacterial activity of ovotransferrin (OVT). Firstly, OVT (purity >90%) was isolated from egg white with a simple and efficient method. After the treatment of ultrasound and glycation, the browning degree of OVT increased with the rising power of ultrasound, while the number of free amino groups obviously decreased to 25.4%. Various spectrum detection showed that the structures of OVT have changed significantly, indicating the tertiary structure became more flexible and looser. The minimal inhibitory concentration of ultrasound glycated OVT were 25.0 and 32.1 μmol/L for E. coli and S. aureus, respectively. In summary, ultrasound-assisted glycation is an effective technique to improve the biological activity of OVT.