Yaw optimization control is recognized as the most effective active wake control strategy for enhancing the overall power generation of wind farms. However, the potential adverse effects of yaw optimization control on the fatigue life of wind turbines remain unclear. This study examined the effect of yaw optimization control on the fatigue life of offshore wind turbines using tower bolts. Initially, the yaw misalignment of the wind turbines was optimized using the open-source FLORIS package to maximize wind farm power generation. Subsequently, the time-varying load of the wind turbines was obtained through the OpenFAST software, using the optimal yaw misalignment, wind speed, and turbulence intensity at the hub height as inputs. The fatigue life of the wind turbines was then calculated by integrating the Schmidt–Neuper engineering stress algorithm, rain flow counting method, and Miner-Palmgren fatigue damage accumulation theory. Finally, a case study of the Horns Rev I large-scale offshore wind farm, comprising 80 wind turbines, revealed that yaw optimization control could significantly enhance the annual power generation of the wind farm by approximately 1.818%. However, optimization comes at the cost of reducing the fatigue life of wind turbine tower bolts by approximately 3–9 years.