This study aimed to screen and identify a novel immune-enhancing peptide and explore the molecular mechanism. Five novel peptides were identified from Agaricus blazei Murrill (ABM), and their secondary structure components consisted of random coil (50.5%), α-helix (28.9%), β-turn (15.6%), and β-sheet (5.0%). A novel peptide (LNEDELRDA) with a molecular weight of 1074.0989 Da could bind with PI3K, AKT, mTOR, IL-6, IL-1β, and TNF-α through hydrogen bonding interactions, and the binding energies were −8.1, −8.3, −7.2, −6.0, −7.4, and −5.8 kcal/mol, respectively. This peptide was synthesized and validated for immune-enhancing ability, showing the strongest immune-enhancing capacity by increasing the cell viability and phagocytic activity of RAW 264.7 macrophages, significantly promoting the production of NO, cytokines TNF-α, IL-1β, and IL-6 in cells, and up-regulating the mRNA and protein expression levels of the PI3K/AKT/mTOR signaling pathway. Our results are the first to reveal that ABM-derived peptide LNEDELRDA could be considered as a promising food-borne immunomodulator that could contribute to enhancing immune function.