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In this paper, the Y2Bi1Fe5O12 films were grown on substituted gadolinium gallium garnet substrate by radio frequency magnetron sputtering and their magnetic properties were investigated. We found that the 20 nm-thick film had the best perpendicular magnetic anisotropy (PMA) under the combined effect of stress-induced anisotropy and surface roughness of the film; the applied magnetic field required for its out-of-plane magnetization saturation was 90 Oe, the remanence ratio was 0.8, the coercive force was 30 Oe, and the damping constant was 3.37 × 10−3. The PMA of the films was reduced due to the relaxation of the stress as the thickness of the film increased, and the easy magnetization axis became in-plane as the thickness reached 40 nm.