A variety of full fat or skim milk samples were taken from the market. The gold standard of exosome extraction， ultracentrifugation （UC）， was used as the control. A variety of chemical pretreatment methods were used to remove the milk protein，and tangential flow ultrafiltration （TFF） was used to further remove the residual milk protein； the primary purification and concentration of milk samples were realized；the bovine milk exosomes were purified by biosize exclusion chromatography （BE-SEC）；nano flow cytometry（NanoFCM） was used to detect the particle size， concentration and purity of the bovine milk exosomes； the morphology of bovine milk exosomes was observed under transmission electron microscope（TEM）；the sizes and purities of the bovine milk exosomes were verified by high performance liquid chromatography（HPLC）； the expressions of specific proteins of bovine milk exosomes were detected by Western blotting method； Gene Ontology （GO） enrichment was used to analyze the candidate target genes of differentially expressed microRNA（miRNA）； DAVID Bioinformatics Resources 6.8 and KOBAS software were used to detect the statistical enrichment of target candidate genes in Kyoto gene and genome encyclopedia （KEGG） pathway； CytoFlex flow cytometry was used to detect the uptake of bovine milk exosomes； flow cytometry was used to detect the apoptotic rate of Caco-2 cells after treated with bovine milk exosomes.

The process development study results showed that the precipitation pretreatment approach with sodium phosphate was superior to all other methods evaluated in regard to feasibility， product quality and reproducibility. TFF method could further remove the residual milk protein and achieved the purpose of liquid concentration， which was effectively linked to the downstream BE-SEC purification method. The exosomes obtained by the three-step process showed the typical disc-shaped or cup-shaped exosomes under TEM.The purity of the sample detected by NanoFCM method was high than that detected by UC，and its partical size was 30－150 nm.The Western blotting results showed that the exosome samples contained CD81， Alix，TSG101 and other exosome specific markers. The SEC-HPLC analysis results showed that the final products of different batches had a single homogeneous peak without free protein pollution. The GO and KEGG database analysis results showed that the milk exosomes were rich in immune related proteins and miRNA related to inflammatory response regulation signaling pathway. The exosomes obtained by the above preparation processes had the ability to penetrate the cell membrane efficiently， and the proportion of exosomes entering the cell within 4 h was 99%.

A three-step purification process is established by chemical precipitation pretreatment， TFF and BE-SEC.The purity and yield of bovine milk exosomes obtained by this method are better than those obtained by gold standard—UC.The physicochemical properties of the samples are in line with the standard of typical bovine milk exosomes， which could penetrate the cell membrane within a few hours， and are rich in miRNA related to immune and inflammatory response regulation.