The supplementation of different ω-6/ω-3 ratio to the diets of the laying hens has been studied to evaluate the effects on performance, egg quality, egg health indices, egg fatty acid profiles, and immune response. One-hundred and twenty, 50-weeks-old hens were divided into three groups fed diets with different ω-6/ω-3 polyunsaturated fatty acids (PUFA) at ratio: 16.7:1, 9.3:1, and 5.5:1, respectively. Each group contained eight replicates of five hens. Hens fed the diet with the highest ω-6/ω-3 ratio had significantly increased weight gain compared to those fed the 5.5:1 and 9.3:1 ω-6/ω-3 ratios. In contrast, hens fed the 9.3:1 ω-6/ω-3 ratios laid significantly more eggs, had increased egg mass, greater livability, and a better FCR than the control group. However, hens fed a ratio of 5.5:1 ω-6/ω-3 PUFA showed improved thrombogenic, atherogenic, hypocholesteremia, and hypocholesteremia/hypercholesteremia indices. In conclusion, laying hens of the 9.3:1 ω-6/ω-3 PUFA group showed improved laying performance, while a ratio of 5.5:1 enriched the ω-3 PUFA in eggs and boosted the immune response of hens.