Ni80Fe20 (NiFe) films have been deposited on self-organized sapphire substrates with periodical surface ripples formed by 2–10 h thermal annealing at 1400°C. By extracting static and dynamic magnetic parameters from magneto-optical Kerr effect (MOKE) loops and ferromagnetic resonance (FMR) spectra, we quantified and analyzed the substrate annealing effects on the induced in-plane uniaxial magnetic anisotropy and ferromagnetic resonance frequency in these films. The study demonstrates that the uniaxial anisotropy of these NiFe films is strongly related to the annealing-time dependent substrate faceting and coarsening mechanisms. The uniaxial anisotropy field Hu changes from ~170 Oe to ~250Oe, and the maximum value of it is obtained in film on 8-h annealed substrate. The ferromagnetic resonance response and high frequency characteristics of films can be modulated by self-organized substrate is also confirmed. When external magnetic field is parallel to the direction of ripple, the ferromagnetic resonance frequency of films grown on the self-organized substrates is increased by ~0.3−1 GHz compared with that of reference film on plane substrate. This work would be helpful to design the high frequency properties of spintronic devices and micro-wave magnetic devices by utilizing self-organized substrates.