Degree of doneness(DOD)is an important factor affecting the palatability and consumer satisfaction of steak. Myofibrillar proteins are important structural proteins, which are closely related to meat quality. Heating is the key technology of meat processing. In recent years, there were lots of articles that reported the effects of thermal treatment on the structural properties of myofibrillar proteins, but few reports on the effects of DOD on its structure. Based on modern infrared spectroscopy, the attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)was combined with two-dimensional correlation spectroscopy, using infrared spectroscopy, second derivative spectroscopy and two-dimensional correlation spectroscopy to track the cooking process of myofibrillar proteins dynamically. The main changes in the protein cooking process were explained by analyzing the trend and regularity of characteristic peaks. The results showed that in the range of 1 700~1 500 cm^-1, DOD had a significant effect on myofibrillar proteins' characteristic absorption peak. As the DOD increased, the intensity change of myofibrillar proteins characteristic peak was divided into three stages: the first stage was the initial stage of heating, and the intensity decreased from control to rare; the second stage was the middle stage of heating, from rare to medium. There was no significant change in peak intensity; the third stage was the late stage, from medium to over-cooked, and the peak intensity decreased significantly. The synchronous spectrum analysis results showed four autopeaks near 1 650, 1 640, 1 556, 1 540 cm^-1, and the cross peaks between the two autopeaks were all positive. The autopeaks intensity analysis results showed that the medium was the turning point of the change of the protein's temperature sensitive region. When proteins were cooked from control to medium, the amide II band was the sensitive region of myofibrillar proteins, while the sensitive region was the α-helix of amide I banded when proteins were cooked from medium to over-cooked. The information of the dynamic changes of myofibrillar proteins molecular structure caused by cooking provided a theoretical basis for the control and optimization of Western steak cooking.