An experiment was conducted to study the effects of flock age and biotin injection at different times of incubation on hatchability and physiological status of newly hatched chicks. One thousand and six hundred and twenty hatching turkey eggs representing two flock ages (32 and 48 wks). All eggs were randomly assigned to three injection time groups, (48 hours, 7 and 24 days of incubation), then each group was divided into six treatments according to the concentration of the vitamin. The first treatment as negative control, the second treatment as a sham-injected control (Dry punch) and the third treatment was injected with 1 ml saline in the air cell through the width end of the egg as sham control. The fourth, fifth and sixth groups were injected with 75, 100 and 125 μg biotin dissolved in 1 ml saline, respectively. Generally, the results indicated that egg fertility rate showed a positive relation with avidin expression in the young-age group than in old-age group. In addition, biotin injection at the late stages of embryogenesis positively affects hatchability, embryo survival percent and body weight of the day-old chicks compared to control and sham groups and influences insulin and thyroid hormones metabolism in post-hatched poults. Based on the findings of the present study injection of turkey eggs with biotin with doses 75 and 100 μg/egg on 24th day of embryogenesis for younger and older hen's markedly improved hatchability and physiological responses for post-hatched chicks.