In order to evaluate the fatigue performance of the steel anchor box (SAB) when its stay cable experiences large-amplitude vortex-induced vibration (VIV) under in-service condition, continuous monitoring was conducted on a long-span cable-stayed bridge. The acceleration of the stay cable undergoing VIV and the stress at the SAB details were measured. The characteristic of stay cable vibration, as well as the stress at the SAB details due to VIV of the stay cable, vehicles loading, and thermal effects, were investigated in both time and frequency domains. Hence the loading mechanisms of VIV, vehicle loading, and thermal effects on the SAB were discussed. Based on the nominal stress method, the fatigue performance of the SAB under the joint action of VIV, vehicle loads and thermal effects were evaluated. The results show that the significant vibration of stay cable, characterized by the high-order multi-mode VIV, dominated by in-plane vibration with peak frequencies between the fifth and the seventeenth modes, occurring within a mean wind speed range of 2 m/s to 9 m/s, with observed maximum in-plane peak acceleration of 25 m/s².Thermal effects significantly contribute to the maximum stress range at the SAB details, although they only generate one stress cycle per day. Compared to the thermal action, the stress range generated by the passage of vehicles is relatively low, but trucks produce a large number of loading cycles. The inertial force generated by VIV of the stay cable applies very low stress to the SAB, making its effects on stress and fatigue negligible. It is concluded that the fatigue evaluation of the steel anchor box should consider the thermal effects. However, even when considering the combined effects of VIV loading, thermal effects and vehicle loading, the fatigue life at the critical details of the SAB—specifically the deck-side welds of the upper and lower plates to the outer web, as well as both weld ends of the bearing plate to the outer web of the steel box girder—exceeds 100 years. Therefore, the fatigue performance of the SAB under in-service conditions meets the bridge design requirements, even with large-amplitude VIV of the stay cable.