Background: Transfer factors (TFs), a novel immunostimulatory reagent, have found use as auxiliary or primary therapy for many diseases. The aim of this study was to explore whether TFs are able to strengthen immune responses of Newcastle Disease (ND) vaccines in broilers.Results: The serum antioxidant status was increased in TF-treatment broilers. TF subcutaneous injection could significantly increase (P < 0.5) the antibody titers at 14 and 21 days of the experiment. Moreover, TF treatment increased development of organs of the immune system, such as spleen, thymus, and bursa of Fabricius through inhibition of apoptosis and promotion of proliferation. Cellular immune responses were found to have higher levels in the groups with TF co-inoculation compared to those groups only treated with the ND vaccine, showing phenomena of higher expressions of interleukin (IL)-2, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ mRNA in the thymus, spleen, and bursa of Fabricius. Moreover, the immunostimulatory effect of TF subcutaneous injection treatment is better than oral and intramuscular injection.Conclusion: our findings suggest that TF treatment can improve antioxidant status and strengthen immune responses to ND vaccine, including antibody production and cellular immunity (lymphocytes proliferation and cytokines production) of the broilers, and the subcutaneous injection of TF is the appropriate inoculation way. Thus, TFs are a potent adjuvant and can serve as a medicine for immunoregulation.