This study investigates the effect of supplementation of Perilla seeds (PS) on the performance, egg quality, blood biochemical parameters, and egg yolk fatty acids composition in the diet of egg-laying chicken. A total of 1600 Lohmann laying hens were randomly assigned to four different groups with 4 replicates each (100 chickens/replicate) and were subjected to varying PS concentrations (PS0, PS6, PS12, and PS18; 0%, 6%, 12%, and 18%, respectively) for four weeks, including an acclimation period of one week. The results showed no significant differences among the groups for average egg weight (P > 0.005). The laying rate (%), feed conversion ratio (FCR) and average feed intake (AFI) decreased significantly for birds fed on 18% PS as compared to the other treatments (P < 0.005). Haugh unit, albumin height, egg-shape index and eggshell thickness among hens fed PS diets were greater averaging 80.53, 7.00, 1.29, 0.34 compared to 76.84, 6.86, 1.25 and 0.32 from Control hen eggs (P < 0.05). Serum analysis showed a trend towards elevated levels of glucose (Glu), total protein (TP) and aspartate aminotransferase (AST) among treatments. Total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) decreased for the birds fed on 6% PS. The fatty acid composition of egg yolk showed a substantial reduction for α-linolenic acid and docosahexaenoic acid increased significantly by the incorporating PS in the diet (P < 0.001). PS incorporation in diets resulted in significant improvements in both performance indicators and greater amounts of α-linolenic acid and DHA in egg yolks. These findings indicate that PS at 6% inclusion has the potential to improve fatty acid profiles of egg yolk without anyadverse effect on performance of egg quality.