To address the limitations of existing studies on harmonic drive (HD) systems, such as the lack of systematic exploration of the influence of flexspline (FS) structural parameters on service lifetime and insufficient parameter optimization strategies, this study establishes a comprehensive research framework integrating finite element (FE) simulation, response surface optimization, and experimental validation. Three key structural parameters of FS, including cup length (CL), cup wall thickness (CT), and tooth width (TW), which were selected as design variables to investigate their effects on the displacement, von Mises stress, and fatigue lifetime of the HD system. A reliable FE model of the HD system was constructed, with mesh independence verified and experimental validation conducted using a servo motor-driven test platform. The results showed that the relative errors between simulated and measured displacement (4.7%) and stress (4.0%) were within the acceptable engineering range (<10%), confirming the model’s reliability. Systematic analysis revealed that the FS lifetime is jointly determined by global stress distribution and structural rigidity, rather than local tooth root stress alone. Increasing cup length reduces tooth root stress but increases cup wall bending stress, which ultimately dominates the fatigue failure process. Based on Box–Behnken Design (BBD) response surface analysis, the optimal parameter combination was determined as CL = 48 mm, TW = 6 mm, and CT = 0.64 mm, achieving the dual optimization of low system stress (77.7 MPa) and long FS lifetime (2950 h). Relative to the baseline control group (CL = 48 mm, CT = 0.46 mm, TW = 6 mm), the optimized configuration reduces system stress by 10.1% and extends fatigue lifetime by 1.7%. This study clarifies the multi-factor coupling mechanism of structural parameters regulating HD system performance, providing a robust theoretical basis and engineering reference for the structural design and performance improvement of HD systems.