Exosomes are small extracellular vesicles found in body fluids. They contain proteins and nucleic acids that reflect the condition of the parent cell. Because of this, they may be useful in colorectal cancer (CRC) diagnostics. In this study we compared three plasma isolation methods: ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial Exo-spin kit. The material obtained with each method was checked for protein content, purity, and vesicle integrity using protein measurements, Western blots, and scanning electron microscopy. From these results SEC was chosen for further use. Exosomes isolated with this method were tested by an enzyme-linked immunosorbent assay (ELISA) for four CRC-related proteins: HSP70, CK19, CA125, and TAG72. Patient samples showed higher levels of HSP70, CK19, and CA125 than controls. TAG72 levels did not differ. In addition, men had more HSP70 than women. These findings show that SEC can be applied for exosome isolation from plasma and that specific proteins detected in exosomes, including HSP70, CK19, and CA125, may serve as promising markers for non-invasive colorectal cancer diagnosis and monitoring.