Sensitive and accurate analysis of low-concentration of tumor-derived exosomes (Exos) in biofluids is essential for noninvasive cancer diagnosis but is still challenging due to the lack of high-sensitive methods with low-cost and easy-operation. Herein, exploiting target Exos as a three-dimensional (3D) track for the first time, we developed a self-serviced-track DNA walker (STDW) for wash-free detection of tumor Exos using exosomal glycoprotein, which was enabled by split aptamer-recognition-initiated autonomous running powered by a catalytic hairpin assembly (CHA). Benefiting from high selectivity and sensitivity of the STDW assay, direct detection of tumor Exos in cell culture medium and serum could also be realized. Furthermore, this method exhibited high accuracy in clinical sample analysis, offering the potential for early cancer diagnosis and postoperative response prediction.