Microalgae (Nannochloropsis sp., NS), with high contents of eicosapentaenoic acid (EPA) and crude protein, may be one of the important n-3 polyunsaturated fatty acid (PUFA) sources and potential protein feed ingredient. The purposes of this study were to enrich yolk with n-3 PUFA by dietary EPA-rich NS supplementation and to evaluate whether it is feasible to partly substitute for soybean meal in laying hens diet. A total of 360 37-wk-old healthy Lohmann Brown laying hens, with similar laying rate and body weight, were randomly allotted to 5 groups (6 replicates, 12 birds/replicate) and fed 5 experimental diets (0, 1, 2, 4, and 8% NS) for 4 wk. The hen performance and egg quality (except yolk color) were not affected (P > 0.05) by the NS supplemental diets. Yolk color score was increased as NS supplementation in diets (P < 0.001), and peaked on about the seventh day in all NS supplemental groups. The concentration of total n-3 PUFA was increased (P < 0.001), while total n-6 PUFA and n-6/n-3 ratio were decreased (P < 0.001) in yolk with increasing NS levels in diets. The 8% NS group had highest docosahexaenoic acid (DHA) and total n-3 PUFA levels, reaching 111.6 mg DHA and 148.6 mg total n-3 PUFA per egg. Maximum DHA, total n-3 PUFA, very long-chain (LC-) n-3 PUFA, and LC-PUFA levels were all observed at day 13 of NS supplementation. In conclusion, dietary NS supplementation enriched yolk with n-3 PUFA (especially DHA) and enhanced yolk color score without adverse effects on performance and egg quality, and indicated the practical feasibility of partial replacement for soybean meal in laying hens diet.