In offshore engineering, fatigue damage caused by flow-induced vibrations (FIVs) presents a considerable and potential threat to flexible cylindrical systems during their service lifetime. Through FIV experiments, this paper estimates and analyzes the fatigue damage of two unequal-diameter cylinders with diameter ratio d/D = 0.5 (where d and D denote the diameter of small and large cylinders) and the center-to-center spacing ratio P/d = 6 (where P denotes the center-to-center distance between two cylinders) through the S–N curve and fatigue damage accumulation methods in the DNV standard. Ten staggered configurations were studied, including two relative positions and five stagger angles (θ = 15°, 30°, 45°, 60°, and 75°). The fatigue damage evaluation results indicate that the FIV fatigue damage is highly affected by the stagger angle and relative position of the two cylinders. The FIV fatigue behavior of the two unequal-diameter cylinders is quite different from that of the two identical cylinders. When the large cylinder is located upstream, its wake shielding effect on the small cylinder is predominant at small stagger angles, so both the IL and CF fatigue damage of the downstream small cylinder is significantly suppressed, especially when Vr ≤ 16.28. But as the stagger angle is increased, the wake-induced flutter can aggravate the fatigue damage of the downstream small cylinder. Moreover, the CF fatigue damage of the large cylinder is decreased at small angles due to the vortex-shedding frequency suppression of the small cylinder and the fatigue damage of the large cylinder in the IL direction is analogous to that of the isolated cylinder and is negligibly affected by the small cylinder. When the small cylinder is located upstream, the IL FIV fatigue damage of the downstream large cylinder is approximately unaffected due to the narrow wake of the small cylinder. However, the CF fatigue damage of the large cylinder is greatly reduced at small stagger angles.