Highland barley wine (HBW) is a well-known grain wine in Qinghai-Tibet Plateau, China and is mainly fermented by local Qu (a traditional starter) with highland barley (Hordeum vulgare, Qingke (Tibetan hulless barley)), and the flavors profiles associated with microbiota succession during HBW fermentation are unrevealed. Hence, high-throughput sequencing (HTS) technology was used to investigate the dynamic changes of microbial community for the duration of the fermentation. In addition, metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC). A total of 66 volatile compounds and 7 organic acids were identified during the traditional brewing process. Results showed that the composition of microbiota varied over the fermentation process. The bacterial genera (relative abundance > 0.1%) decreased from 13 at 0 h to 4 encompassing Leuconostoc (13.53%) and Acetobacter (74.60%) after 48 h fermentation, whilst the structure of fungal community was more uniform in comparison with bacteria, as Rhizopus and Saccharomyces were predominant throughout the fermentation. Furthermore, the correlations between microbiota and the detected compounds were also explored, which highlighted that three bacterial genera, including Acetobacter, Leuconostoc, Bacillus and one fungal genus Rhizopus were significantly correlated with main flavours compounds (|r| > 0.7, FDR < 0.01). To conclude, the detailed information provided by this study offer screening strategies of beneficial bacterial and fungal strains to improve the quality of HBW.