Ag-Co films with ultra-high resistivity were prepared on polyimide by magnetron sputtering. The effect of Co content and annealing temperatures on the resistivity and microstructure of Ag-Co films has been thoroughly investigated and the relation between resistivity and microstructure has been discussed. Results show that thicker Ag-Co films without annealing present lower resistivity due to better crystallinity. However, thin Ag-Co films (≤21 nm) annealed at 360 °C present ultra-high film resistivity because of the formation of diffusion pits on the film surface which blocks the transmission of electrons in films to increase film resistivity. Inversely, the resistivity of thick Ag-Co films (≥45 nm) annealed at 360 °C is much less than that annealed at lower than 260 °C owing to no diffusion pits. Furthermore, the addition of Co inhibits the growth of Ag grains and limits the migration of electrons in Ag-Co films further, also resulting in the increase of Ag-Co films’ resistivity.