The invention relates to a method for separating exosome from a human placenta-derived mesenchymal stem cell source. The method comprises the following steps: when the P2-P3 placenta-derived mesenchymal stem cell convergence rate reaches 85-90%, obtaining a cell culture supernate, carrying out membrane filtration, and collecting the filtrate; centrifugating the filtrate, collecting the centrifugal supernate, adding a 10w / v%-12w / v% polyethyleneglycol culture medium supernate solution into the centrifugal supernate according to the volume ratio of 1:1, sufficiently and uniformly mixing, and carrying out secondary centrifugation, wherein the finally obtained precipitate is the exosome. The method is convenient for taking materials, is easy for enrichment amplification culture, reutilizes the P2-P3 placenta-derived mesenchymal stem cell culture medium supernatant to obtain the exosome, and does not refer to the problem of medical ethics. In the exosome separation process, membrane filtration, centrifugation, polyethyleneglycol solution addition and other means are adopted to effectively increase the exosome enrichment, so the method has the advantages of short time consumption and low cost.