In this work, we have designed a template-free multiple signal amplification method for the highly sensitive detection of cancer cell-derived exosomes. In this design, DNase I serves as a bridge to link the DNA-based amplification approach and terminal deoxynucleotidyl transferase (TdT)-mediated polymerization reaction. Consequently, a detection limit of 10 particles per μL can be achieved, while a complex nucleic acid sequence design can be avoided. This method also exhibits good performance in a complicated matrix and enables the differentiation of healthy individuals from colorectal cancer (CRC) patients.