In this paper, the high-throughput sequencing and cultured methods were combined for analyzing the diversity of microorganism in Highland barley Qu and determining the dominant strain.The results suggested that the predominant fungi were Rhizopus, Aspergillus, Ophiocordyceps and Saccharomyces, whereas Rhizopus oryzae was the major fungus and the main microorganism for saccharification.Moreover, the predominant bacteria were Kocuria, Macrococcus, Bacillus and Micrococcus.In addition, the results of the optimal Koji-making condition with Rhizopus oryzae showed that the enzymes activity of saccharifying enzyme and liquifying enzyme were up to 1 246.9 U/g and 61.4 U/g when it was cultured at 30 ℃ for 72 h with raw material ratio of 2∶1, liquid ratio of 40%, and inoculum of 3.5%, which were 9.2 times and 2.4 times higher than that of originally Qu, respectively.