Dairy calf gut health is linked with development and future production. Fecal extracellular vesicles (fEV) have emerged as a noninvasive tool in elucidating gut physiology and pathophysiology. Because feces is a complex matrix, the enrichment of extracellular vesicles (EV) from ruminant or preruminant feces is difficult. Nevertheless, if enriched, they have great potential as a gut health diagnostic and monitoring tool in dairy calves. Therefore, this study aimed to devise a protocol to enrich and characterize fEV from preweaning calves. We developed an fEV enrichment method by combination of differential centrifugation and double size exclusion chromatography and then characterized the fEV from the healthy calves. The study also assessed sample storage conditions, and the results indicated that storing preprocessed fecal samples at -80°C effectively preserves EV without introducing additional nanoparticles. Finally, fEV from 10-d-old healthy and Cryptosporidium spp.-positive calves were enriched, and a comparative analysis of fEV characteristics between the 2 groups was performed. Characterization results on EV specific protein biomarkers, size profile, total protein content, zeta potential, and morphology clearly established the enrichment of fEV with the developed protocol. The fEV analysis for calves positive and negative for Cryptosporidium spp. revealed a significant decrease in average nanoparticle size and zeta potential values in Cryptosporidium spp.-infected calves. Furthermore, the enriched fEV carried protein and nucleic acid cargo which could be further analyzed for other biomarkers to predict the gut physiology and pathophysiology of calves. In conclusion, our study has successfully optimized a protocol to enrich high purity grade EV from calf feces and displayed potential diagnostic application as a noninvasive tool.