Microbial diversity in kefir grains is responsible for the production of goat milk kefir with unique peptides composition and volatile profile. High-throughput sequencing technique was used to analyze bacterial and fungal diversity of three different kefir grains which were originated from China, Europe Germany and United States. Peptides and volatile profile in goat milk kefir were determined by proteomic platform and Gas Chromatography-Ion Mobility Spectrometry, respectively. Clustering analysis indicated that the different content of Lactobacillus genera in different kefir grains was highly associated with the proteolytic ability in goat milk kefir. Contents of volatile compounds in goat milk kefir were also correlated to the bacteria and fungi in kefir grains (especially for Lactobacillus spp. and Saccharomyces spp.). The innovation of this study was to find a new way in exploration of the correlation of microbiota in kefir grains with the proteolytic ability and volatile profile of goat milk kefir.