The effects of delta-aminolevulinic acid (DALA) or chito-oligosaccharide (COS) in layer diets on production performance, egg quality and hematological characteristics were investigated in this 8-week trial. Two hundred and seventy 27-week-old (Hy-Line brown) layers were randomly assigned to 5 treatments with 9 replications per treatment. Dietary treatments were i) Control (basal diet); ii) DALA1 (basal diet+DALA 2 mg/kg); iii) DALA2 (basal diet+DALA 4 mg/kg); iv) COS1 (basal diet+COS 100 mg/kg) and v) COS2 (basal diet+COS 200 mg/kg). In this study, no significant difference was observed in feed intake and egg shell quality (eggshell breaking strength and egg shell thickness) among treatments. The inclusion of DALA increased egg production during the first 4 weeks. Egg weight was increased in both DALA (quadratic, p<0.05) and COS (linear, p<0.05) treatments compared with the control treatment. Laying hens fed the DALA treatment had an increased yolk color unit (quadratic, p<0.05) and haugh unit (linear, p<0.05) compared with the control group. Similarly, these characteristics were also affected by COS treatments, with both values being linearly increased (p<0.05) in COS treatments compared with the control treatment. Additionally, birds fed DALA treatments significantly increased (quadratic, p<0.05) the number of RBC, WBC and lymphocytes compared with the control treatment. Dietary DALA supplementation linearly increased (p<0.05) the serum iron concentration at the end of the 8th week. The inclusion of COS increased (linear, p<0.05) the concentration of RBC, WBC and lymphocytes compared with the control treatment. In conclusion, dietary DALA at the lower dosage (2 mg/kg) could exert better effects in laying hens than higher dosage (4 mg/kg). Birds fed DALA supplemented diet had an increased iron availability, egg weight, eggshell quality and immunity. Moreover, the inclusion of COS (200 mg/kg) can increase egg weight, eggshell quality and immunity in laying hens. Therefore, both the utilization of COS and DALA could be considered as a new strategy for optimizing egg quality and health condition of laying hens.