A study was conducted to investigate the effects of 3 different rearing systems [free range (FR), semi-intensive (SI) and confinement (CF)] on blood biochemical profile and immune response in 4 varieties of Aseel chicken [Lakha (LK), Mushki (MS), Peshawari (PW), and Sindhi (SN)] for 10 wk duration (7 to 16 wk). At the age of 6 wk, in total, 180 cockerels were assigned to 12 treatment groups, 3 (rearing system) × 4 (Aseel chicken variety) factorial arrangement in 7 randomized complete blocks, replicated 3 times with 5 birds in each replicate (45 birds of each variety; 60 birds in each rearing system; 36 total replicates). Blood samples were collected through brachial vein at the end of wk 16. After laboratory analysis, the recorded data for blood biochemical profile and immune response were analyzed by using 2-way ANOVA under factorial arrangement. The results showed higher (P<0.05) plasma glucose and total protein in birds under CF. Titer against Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) was found to be greater (P<0.05) in SI and FR, respectively. Peshawari birds indicated higher (P<0.05) concentration of glucose, total protein, albumin, uric acid, creatinine, and titers to NDV and IBV. Birds of LK and SN varieties also indicated maximum antibody titer against NDV and IBV, respectively. Cholesterol level was found to be greater (P<0.05) in birds of LK and SN. Interaction of SN with FR revealed maximum (P<0.05) cholesterol whereas interaction of PW with SI indicated maximum antibody titer against NDV. The results highlight that CF rearing system expediently affects glucose and total protein levels in birds; SI and FR confer maximum antibody titers to NDV and IBV. Birds of PW variety indicated higher glucose, total protein, albumin, uric acid and creatinine, the lowest cholesterol under FR and the enhanced antibody titer against NDV and IBV.