Vaccination is the most effective mean of preventing, controlling, and even eradicating infectious diseases. Poultry are vaccinated through various routes including eye/nose drops, drinking water, vent brush, or injections. The prolonged suppression effect of maternal antibodies on humoral immune response of newly hatched chicks to active immunization has been reported, while the effect of vaccination route on this suppression still unclear. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP or non-specific IgY antibodies were transferred by yolk sac inoculation to newly hatched chicks (chicks of maternal and no-maternal antibodies), and they were immunized with DNP-KLH via intrabursal route at 0.1, 1 and 10 mg/kg body weight (BW) and with 1 mg/kg BW intraperitoneally at 1 and 4 weeks of age. Concentration of anti-DNP antibodies in serum samples of these chicks was measured by using Enzyme- linked immunosorbent assay (ELISA). The immune response to intrabursal immunization was higher in chicks of no-maternal antibodies than that of chicks of maternal antibodies at 5 weeks of age. Intrabursal immunization showed higher response than intraperitoneal one at the same dose. These results confirmed that the immune suppressive effect of maternal antibodies on the immune response of the newly hatched chicks was antigen specific and depends on the ratio of antigen/maternal antibody at the time of immunization. Furthermore, intrabursal vaccination showed promising results than intraperitoneal vaccination at the same dose.