The present study was designed to investigate the impact of in-ovo injection of folic acid and glucose on hatching eggs from 55 weeks old broiler breeders. A total number of 900 hatching eggs were collected from Arbor Acres broiler breeders, then, eggs were divided into 6 groups including 1) Negative Control (non-injected, NC), 2) Dry Punch Control (pricked without injecting any solution, DPC), 3) Positive Control (eggs were injected with 0.5 mL normal saline, PC), 4) Folic Acid group (eggs were injected with 0.2 mg/ egg folic acid, FA), 5) Glucose group (eggs were injected with 125 mg/ egg glucose, Glu), and 6) Folic Acid with Glucose group (eggs were injected with 0.2 mg folic acid with 125 mg/ egg glucose, FA+Glu). Each treatment was divided into five replicates of 30 eggs each. Eggs were injected into the albumen under the air sac. After in-ovo injection, the eggs were stored for four days before hatching. After hatching, the chickens were reared in groups according to the treatments. All treatments were divided into 10 replications of 9 chickens in each. In-ovo injection with folic acid decreased the albumen pH significantly to 9.19 after 4 days of injection, while the negative control was 9.43. Hatching quality was severely affected by all in-ovo injection treatments, but no significant differences were found between the treatment groups concerning the hatchability of fertile eggs. Injection treatments had no significant effect on the growth rate or the production number in any of the weeks. Injection of folic acid and (FA+Glu) significantly increased chickens’ body weight at two and four weeks of age. Also, the dressing percentage when using folic acid and (FA+Glu) was significantly increased to 72.1% and 72.5%, respectively, compared to the positive control group (68.3%). In conclusion, our data suggested that in-ovo injection with a mixture of folic acid and glucose (0.2 mg folic acid+ 125 mg/ egg glucose) could be used to enhance carcass characteristics. Further studies should be conducted to find the effects of in-ovo injection folic acid and glucose on different incubation days and at different sites of injection.