A novel cascade signal amplification based on tyramide signal amplification (TSA) and surface-initiated enzymatic polymerization (SIEP) was first reported for the sensitive and template-free detection of colorectal cancer (CRC) exosomes. This assay exhibited 20.9-fold signal amplification with a low detection limit of 12.8 particles per μL. Furthermore, accurate and reproducible results were obtained for detecting exosomes in serum samples, suggesting its potential application in exosomes analysis and clinical diagnostics.