Effects of dietary copper-loaded chitosan nanoparticle (CNP-Cu) supplementation on growth performance, hematological and immunological characteristics, and the cecal microbiota in broilers were investigated. Three hundred healthy Avian × Avian (1-d-old) broilers were randomly assigned into 5 dietary groups (20 birds per replicate with 3 replicates per group). Birds were fed with 0 (the control group), 50, 100, 150 mg/kg of CNP-Cu and 50 mg/kg chlorotetracycline (CTC, a positive control group) for 42 d. Results indicated that supplemental CNP-Cu could improve growth performance, affect the immune system, enhance protein synthesis, and be beneficial to cecal microbiota of Avian broilers, especially the dietary supplementation with 100 mg/kg of CNP-Cu. Supplementation with 100 mg/kg of CNP-Cu increased the average daily gain(P < 0.05) and the contents of IgA (P < 0.01), IgG (P < 0.01), IgM (P < 0.01), complement C3 (P < 0.05), and complement C4 (P < 0.05). Thymus, spleen, and bursa of Fabricus indexes and the populations of Lactobacillus and Bifidobacterium in cecal digesta were increased (P < 0.05) by 100 mg/kg of CNP-Cu supplementation, and the population of coliforms was decreased (P < 0.05). Dietary supplementation with 100 mg/kg of CNP-Cu increased (P < 0.05) concentrations of serum total protein and albumin, and decreased (P < 0.05) the content of urea nitrogen in serum. Effects of dietary supplementation with 100 mg/kg of CNP-Cu were similar to 50 mg/kg of CTC supplementation. These results may indicate that CNP-Cu could be a new substitute for CTC in dietary supplementation.