Exosomes are nanoscale (30-150 nm) biological vesicles that are actively released from living cells and circulating into all body fluids. Recently, exosomes in cerebrospinal fluid (CSF) have been recognized as promising biomarkers for central nervous system (CNS) neoplasms. In this study, we report a label-free method that can be used to rapidly isolate exosomes from CSF for proteomic analysis. Compared to ultracentrifugation and polyethylene glycol-based precipitation, our method isolates exosomes from 2 mL of CSF within 10 min, which is 18 times and 72 times shorter, respectively; the yield was increased by 4.47 times and 2.09 times and the purity was increased by 4.54 times and 9.76 times, respectively. The proteomic analysis further revealed that the exosomes isolated by our method identified more exosome-related proteins, which may reflect the physiological status of diseases for exosome-based diagnosis. Therefore, the effective isolation of pure exosomes from CSF samples for protein analysis will benefit the downstream analysis and clinical translation of exosomes, thus promoting the early diagnosis of CNS neoplasms.