An experiment was conducted to evaluate the effect of injecting hatching eggs with different concentrations of biotin at different times on the quality and physiological characteristics of hatched chicks. Six hundred eggs hatching (Arbo Acres) were divided into five groups (120 eggs for each group). Treatment groups were distributed as follows: To control group (without injection), T1 injected by 58 µg biotin at age of zero, T2 injected by 68 µg biotin at age of zero, T3 injected by 58 µg biotin at age of 18 days of incubation and T4 injected by 68 µg biotin at age of 18 days of incubation. The data showed that biotin injection contain 58 µg at age of zero lead to significantly increase (P<0.05) in body weight of the day-old chicks, length and width of breast, length of back, length of thigh, relative weight of the bursa of fabricia, spleen and glandular stomach. Significantly increase (P<0.05) in packed cell volume (PCV), total white blood cells count (WBC), concentrations of glucose, cholesterol, total protein, percentage of lymphocytes and significant decrease (P<0.05) in the percentage of hetrophiles and the ratio of H/L.