Currently, numerous studies are conducted on the fatigue fracture performance of concrete, considering various factors such as material ratio, material content, and external environment. However, very few studies consider the impact of casting defects on the fatigue fracture performance of concrete. Cold joints, one of the typical casting defects, are weak joints caused by the discontinuous pouring of concrete structures. To investigate the fatigue fracture performance of concrete with cold joint defects, this study conducted fracture experiments using static load and interface nanoindentation experiments on 12 concrete experimental components with cold joints to analyze the fracture performance and cold joint interface characteristics. Subsequently, this study conducted fatigue fracture experiments on 144 experimental components with cold joints to analyze the fracture characteristics and fatigue life under low stress and low stress amplitude fatigue loads. The results indicate that the peak load, initiation fracture toughness, and unstable fracture toughness of concrete members with cold joints decrease with the extension of the pouring interval of cold joints. The thickness of the cold joint interface increases with the extension of the pouring interval, while the strength decreases with the extension of the pouring interval. The fatigue life of experimental components with cold joints reduces with stress and stress amplitude raising. Under the same initial stress and stress amplitude, the fatigue life of concrete members with cold joints decreases as the pouring interval extends, and the bearing performance deteriorates further after the same number of cyclic loading. Fatigue loads with low stress and low stress amplitudes can lead to the fatigue fracture of experimental components with cold joints. The research findings can serve as a foundation for the study of damage and cracking in lining with cold joints under aerodynamic fatigue loads in high-speed railway tunnels.