The intestine is a site of diverse functions including digestion, nutrient absorption, immune surveillance, and microbial symbiosis. Intestinal microRNAs (miRNAs) are detectable in faeces and regulate barrier integrity, host-microbe interactions and the immune response, potentially offering valuable non-invasive tools to study intestinal health. However, current experimental methods are suboptimal and heterogeneity in study design limits the utility of faecal miRNA data. Here, we develop an optimised protocol for faecal miRNA detection and report a reproducible murine faecal miRNA profile in healthy mice. We use this pipeline to study faecal miRNAs during infection with the gastrointestinal helminth, Trichuris muris, revealing roles for miRNAs in fibrosis and wound healing. Intestinal fibrosis was confirmed in vivo using Hyperion® imaging mass cytometry, demonstrating the efficacy of this approach. Further applications of this optimised pipeline to study host-microbe interactions and intestinal disease will enable the generation of hypotheses and therapeutic strategies in diverse contexts.