Ganoderma sinense is a traditional and protein-rich edible fungus with outstanding immunoregulatory activity. However, the mechanism through which G. sinense protein hydrolysates and peptides exert their immunomodulatory effects is unclear. The objective of this study was to prepare and isolate immunologically active peptides from G. sinense protein hydrolysates (GSP) and to investigate their impact on the activation of macrophages. G. sinense peptides with low molecular weights (< 1 kDa, 87.76%) markedly promoted RAW264.7 cell proliferation; increased their phagocytic capacity, nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and IL-6 secretion and reactive oxygen species (ROS) production; and upregulated Tlr2 mRNA expression. In addition, GSP promoted nuclear factor kappa-B (NF-κB) activation and translocation through the upregulation of p65 and p-p65 protein expression. Virtual molecular screening technology revealed that compared with other peptides, the SFAGNIPVNR, YGDAFIR and TVSYLPAPQR peptides derived from GSP showed stronger binding affinities. Interaction site map analysis indicated that the SFAGNIPVNR and YGDAFIR peptides formed stable hydrogen bonds with toll-like receptor 2 (TLR2). Together, these results suggested that G. sinense peptides can serve as important ingredients in nutraceuticals or functional foods.