A wide range of medicinal values and good health effects characterizes pseudognaphalium affine (D.Don) Anderb. PHP, as a polysaccharide fraction, was isolated and purified from Pseudognaphalium affine (D.Don) Anderb. This study evaluated PHP's structural characteristics in terms of its molecular weight, functional groups, and monosaccharide content. After dextrose sodium sulfate (DSS)-induced ulcerative colitis (UC) in zebrafish, the anti-inflammatory efficacy of PHP against macrophage RAW264.7 in vitro was assessed, as well as its impact on pathological alterations, oxidative stress state, and expression of inflammation-related genes. According to the data, PHP is a neutral heteropolysaccharide comprising nine monosaccharides. It markedly decreased the secretion of pro-inflammatory cytokines (TNF-alpha, IL-6) and altered the oxidative stress status of RAW264.7 cells. It increased oxidative stress markers and decreased the clinical signs of zebrafish colitis (CAT, SOD). PHP concurrently controlled the expression of genes linked to the NF-kappaB and MyD88 signaling pathways. Overall, PHP showed striking alleviation of ulcerative colitis in zebrafish, which can effectively facilitate the development of adjuvants and medicinal and edible plants to treat ulcerative colitis.