Steel structure flange connections are extensively employed in structural nodes due to their superior mechanical properties. This study combines fatigue testing and theoretical methods to investigate the fatigue performance of high-strength bolts in flange connections under actual gradient descent loads and provide fatigue design methods. Initially, fatigue tests were conducted on two sets of high-strength bolts under a gradient descent loading mode, yielding a total of 11 sets of fatigue data. Subsequently, the stress–life (S-N) curve was plotted using a cumulative damage model combined with an equivalent constant amplitude stress method, and the results were compared with existing fatigue design specifications. Additionally, digital cameras and electron microscopes were utilized to capture fatigue fracture images of the high-strength bolts, allowing a detailed investigation into the mechanisms underlying bolt fatigue fractures. The results indicate that the allowable stress amplitudes for the two sets of high-strength bolts, corresponding to a fatigue life threshold of 2 million cycles, were 144.211 MPa and 130.316 MPa, respectively—both of which exceed the values specified in current fatigue design codes. Moreover, finite element simulations revealed that the most pronounced stress concentration occurs at the first thread where the bolt and nut interface, which is identified as the critical location for fatigue fracture in bolts. The allowable stress and fatigue calculation method of bolts obtained in this study will provide a reference for flange node design