The present study was performed to investigate the effect of oral administration of β-glucan (G70), a product obtained from the cell wall of yeast, on Newcastle disease virus (NDV)-specific hemagglutination inhibition (HI) titers, lymphocyte proliferation, and the role of T lymphocyte subpopulations in chickens treated with live NDV vaccine. In addition, the influence of β-glucan on splenic gene expression was investigated by transcriptome sequencing. The results revealed that the supplementation of β-glucan boosted the titer of serum NDV HI increased the NDV stimulation index of lymphocytes in peripheral blood and intestinal tract, and promoted the differentiation of T lymphocytes into CD4T cells. The RNA sequencing (RNA-seq) analysis demonstrated that G70 upregulated the mRNA expressions related to G-protein coupled receptor and MHC class I polypeptide, and downregulated the mRNA expressions related to cathelicidin and beta-defensin. The immunomodulatory effect of G70 might function through mitogen-activated protein kinase signaling pathway. To sum up, G70 could boost the immunological efficacy of live NDV vaccine in chickens and could be applied as a potential adjuvant candidate in the poultry industry.