Ovotransferrin (OVT) is very rich in nutritional value and possesses a variety of biological activities. However, there is a lack of suitable OVT extraction methods that are simple and suitable for large-scale production. For this reason, this study explored a new method of ovalbumin OVT extraction based on mesophilic treatment. The effects of different heat treatment conditions on the physicochemical properties and bioactivities of the prepared OVT and their influence mechanisms were investigated. The results showed that OVT could be efficiently extracted from egg white by moderate heat treatment. Based on single factor experiments, response surface methodology was used to determine the effects of heat treatment time, temperature and pH on the extraction rate of OVT. The yield was 93.65 ± 0.53% under the optimal extraction conditions (62.5 °C, 75 min, pH 8). SDS-PAGE and FT-IR showed that changes in the influencing factors during heating had different effects on OVT. In addition, different extraction parameters had different effects on the iron-binding and antioxidant capacities of OVT. This study provides a fast and efficient preparation method for OVT from egg white, which lays the foundation for the wide application of OVT.