The treatment of colorectal cancer (CRC) has improved during the last decades, but methods for crucial early diagnosis are yet to be developed. The influence of the tumour microenvironment on liquid biopsies for early cancer diagnostics are gaining growing interest, especially with emphasis on exosomes (EXO), a subgroup of extracellular vesicles (EVs). In this study, we established paired cancer-associated (CAFs) and normal fibroblasts (NF) from 13 CRC patients and investigated activation status-related protein abundance in derived EXOs. Immunohistochemical staining of matched patient tissue was performed and an independent test cohort of CRC patient plasma-derived EXOs was assessed by ELISA. A total of 11 differentially abundant EV proteins were identified between NFs and CAFs. In plasma EXOs, the CAF-EXO enriched protein EDIL3 was elevated, while the NF-EXO enriched protein QSOX1 was diminished compared to whole plasma. Both markers were significantly reduced in patient-matched CRC tissue compared to healthy colon tissue. In an independent test cohort, a significantly reduced protein abundance of QSOX1 was observed in plasma EXOs from CRC patients compared to controls and diagnostic ROC curve analysis revealed an AUC of 0.904. In conclusion, EXO-associated QSOX1 is a promising novel marker for early diagnosis and non-invasive risk stratification in CRC.