Researchers are trying to boost the immune system of poultry birds through the use of natural immune-stimulants including bee venom. There is a scarcity in literature about the use of bee venom for in ovo injection studies. The aim of this study is to investigate the effect of in ovo injection of two levels of freeze-dried bee venom (BV) (10 and 20 µg/egg) into eggs of Alexandria breeder hens at 18-day of incubation on the hatchability percentage, hatching weight per egg weight, and immune response of the young chickens. The doses of BV used in the current work were based on a pilot trial obtained directly before the main experiment of this work. A total number of 900 fertile eggs at day 18 of incubation were used in this study. Eggs were weighed and randomly allocated into three experimental groups as: control (C), low dose of BV and high dose of BV with 300 eggs each. Results showed that in-ovo injection with 10 µg/egg BV significantly increased hatchability percentage (89.45%) over the control (87.5%) and 20 µg/egg BV (77.73%). The highest relative chick weight was observed with eggs of control group followed by lower and higher dose of BV groups. The increase of dose has reduction effect on the relative chick weight. The total protein and albumin values decrease significantly (P≤0.001) in 10 and 20 µg BV/egg groups compared to control group. Results for triiodothyronine (T3), and thyroxine (T4) concentrations of chick's blood at hatch showed that in-ovo injection of BV decrease significantly both of T3 and T4 concentrations in Alexandria chick serum at hatch. In addition, results of showed that the spleenrelative weight increased significantly in 20 BV dose in-ovo group compared to control group. Also, the same trend was observed with bursa relative weight compared to the control group. In conclusion, the in-ovo injection with 10 µg BV/egg resulted in better hatchability percentage with Alexandria breeder eggs.