Royal jelly renowned for its robust nutritional, functional, and biological properties, is a pivotal product derived from honeybees. The purpose of this investigation was to assess the theory that in ovo injection of freeze-dried royal jelly (FDRJ) solutions at varying concentrations can influence the hatchability, blood properties and hatching characteristics of day-old chicks. A total of 480 eggs (54.81 ± 0.187 g) were allocated into four experimental groups: negative control (NC), without injection, positive control (PC), administered with regular saline, a low FDRJ dose group (9 mg/egg), and a high FDRJ dose group (18 mg/egg). The in ovo injections were administered on Day 18 of incubation, and the experiment was subsequently continued until the incubation period concluded at 21 days. Results revealed that the lower FDRJ dose (9 mg/egg) significantly improved hatchability percentages compared to other treatments. Conversely, the higher FDRJ dose (18 mg/egg) and control groups (NC and PC) resulted in significantly higher chick yield percentages than the lower FDRJ and PC groups. The NC group showed the supreme yolk sac (YS) percentage, whereas the yolk-free body mass (YFBM) percentages displayed an inverse trend. Furthermore, the in ovo FDRJ injection did not affect haematological values or the relative organ weight of day-old chicks. In conclusion, in ovo FDRJ injection demonstrated beneficial effects on hatchability and chick weight, as evidenced by the studied parameters.