Objective: To establish a method for the isolation exosome from serum by polyethylene glycol (PEG). Methods: The ultracentrifugation, commercial EXOQuick precipitation kit and PEG8000 precipitation method were used to isolate exosomes from blood serum of the patients with breast cancer and benign breast lesions and the healthy individuals, respectively. The morphology and particle size of exosomes were measured by transmission electron microscopy and NanoSight NS90 method, respectively. The expression level of the surface marker CD9 in serum exosomes was determined by Western blotting. The expression level of microRNA-21 (miR-21) in serum samples and serum exosomes was detected by real-Time fluorescent quantitative PCR. Results: A method of isolating exosomes by PEG was established successfully. In terms of particle size distribution and CD9 concentration on the surface of exosomes, the performance of PEG8000 precipitation method was superior to ultracentrifugation, and equivalent to the commercial EXOQuick kit. Compared with breast benign lesions, the level of miR-21 was upregulated by 2.68 fold (P =0.024) in serum exosomes isolated from malignant breast cancer patients by using PEG8000 precipitation method. However, the miR-21 expression level was almost equal in serum samples from the patients with breast benign lesions and breast cancer (P =0.373). Conclusion: PEG8000 precipitation method for enriching exosomes is easy to operate, short of time and low in cost. The detection efficiency of miRNA expression in exosomes can be increased significantly than that in serum samples.