This study was performed to investigate and compare the effects of using Artemisia annua leaves, probiotic, and organic acid on the performance, egg quality, blood biochemistry, and antioxidant status of laying hens. In total, 288 Hy-Line W-36 commercial layers (32 weeks old) were divided into six groups with six replicates per group (eight birds per replicate) and were fed one of six experimental diets. The hens were fed either a corn-soybean meal basal diet (control) or the basal diet supplemented with 2.5% A. annua leaves (AA1), 5% A. annua leaves (AA2), 7.5% A. annua leaves (AA3), 0.1% probiotic (Pro), and 0.005% organic acid (Org), respectively. The experiment lasted 10 weeks. Results showed that there were differences in the feed conversion ratio (FCR) among experimental groups (P<0.05). The highest yolk color index and shell thickness were observed in hens fed AA3 and AA2 diets (P<0.05). Egg yolk cholesterol was decreased (P<0.01) by the diet containing AA3 and Pro compared to the other groups. The atherogenic index was lower (P<0.01) in the plasma of hens fed AA3 than those in other groups. The glutathione peroxidase activity (GSH-Px) and malondialdehyde (MDA) contents in layers fed AA3 were lower and higher (P<0.05), respectively, than in layers fed the other diets. Moreover, the concentration of plasma cholesterol was decreased (P<0.05) in layers fed AA3 and Pro. In conclusion, feeding laying hens with A. annua leaves positively influenced the plasma antioxidant status, and the dietary inclusion of A. annua leaves plus a probiotic significantly decreased the egg yolk cholesterol, with no adverse effect on the egg productive traits.