The conformation changes of Apo-Ovotransferrin and Holo-Ovotransferrin were studied with the heat treatment 25~95 ℃ by using Fourier transform infrared spectroscopy (FTIR) and two-dimensional correlation spectroscopy analyzer. The results of one-dimensional infrared spectroscopy showed that with the increase in temperature, the peak at 3 300 cm-1 of Apo-Ovotransferrin shifted more than that of Holo-Ovotransferrin. The peak at 3 300 cm-1 derived from stretching vibrations of N—H and O—H indicates that iron-binding enhanced the role of hydrogen bonds and resistance to heat. The changing order of the secondary structure of ovotransferrin was determined by analyzing two-dimensional infrared spectra, witch is β-sheet>amideⅡ>—CH2— bending vibration. In addition, it was found that the cross-peaks at 1 652 and 1 688 cm-1 are different in synchronous and asynchronous counter maps by comparing Apo-Ovotransferrin with Holo-Ovotransferrin. It was suggested that the temperature made less impact on the α-helix in Holo-Ovotransferrin than on that in Apo-Ovotransferrin, however, the β-turn in Holo-Ovotransferrin was more sensitive to temperature.