In order to improve the mechanical response and fatigue life of the flexible spline (FS) during the assembly and meshing of the rigid-flexible wheel gear of the harmonic drive, an assembly and meshing analysis method of rigid-flexible gear system based on rigid-flexible coupling and transient dynamic analysis theory was proposed. Stress distribution and fatigue failure position of FS were obtained more accurately and reasonably. The sensitivity of the structure parameters of FS and material property to its fatigue life indicates that the key parameters of the fatigue design of FS, and the fatigue life size effect of FS which has sensitive range, are also noted. In addition, the fatigue life model of FS was established. The results show that the mechanical and fatigue life analysis of the rigid-flexible gear system of the harmonic drive cannot ignore the influence of the assembly and meshing process. The design of flexible wheel should avoid the sensitive range of fatigue life size effect to improve the service life of harmonic drive.