The aim of this study was to investigate the role of Dendrobium officinale crude polysaccharides (DOPS) and D. officinale purified polysaccharides (DOPS100) in attenuating immune dysfunction in subfertile mice. The results of the study revealed several noteworthy findings. First, DOPS and DOPS100 treatments led to significant improvement in weight gain and reversal of fatigue-related behaviors compared to the normal group. Second, both DOPS and DOPS100 showed effectiveness in reducing immune organ swelling, modulating immunoglobulin A (IgA) and immunoglobulin M (IgM) levels, and restoring complement (C3c and C4) levels. In addition, they demonstrated a significant ability to enhance the integrity of the intestinal mechanical barrier by differentially upregulating the tight junction proteins Occludin and Zonula occludens 1 (ZO-1). In addition, it was found that DOPS100 specifically enhanced the CD4+-T helper 17 cell (Th17)/ regulatory T cell (Treg) immune axis in the gut, as evidenced by increased expression of forkhead box protein 3 (Foxp3) as well as decreased expression of retinoic acid receptor-related orphan receptor γt (RORγt), and further modulation of interleukin 10 (IL-10), interleukin 22 (IL-22), and interleukin 17A (IL-17A) expression levels of inflammatory factors. These findings collectively suggest that DOPS100 holds significant potential in improving sub-healthy status by repairing the intestinal barrier, restoring local immune homeostasis, and activating the intestinal immune regulatory network. The study's outcomes provide valuable insights into the therapeutic implications of DOPS and DOPS100 in addressing immune dysfunction in sub-healthy conditions.