In this study， we isolated the flavonoid 3Oglucosyltransferase gene named HvtUF3GT from ‘Heilaoya’ grain， and obtained a 1 449 bp sequence. HvtUF3GT gene contained a 1 434 bp open reading frame (ORF)， encoding 478 amino acids. The theoretical isoelectric point (pI) was 5.45 and the predicted protein molecular weight was 52 912.58 Da. Protein sequence analysis showed that HvtUF3GT was a hydrophilic unstable acidic protein containing a typical 44 amino acids PSPG box at the Cterminus and a βαβαβ Rossmann folded structure in the spatial structure. HvtUF3GT was a member of UDP glycosyltransferase_GTB _type superfamilies. Phylogenetic tree analysis showed that HvtUF3GT and the glycosyltransferase of Trib. Triticeae such as Hordeum vulgare， Triticum urartu， Aegilops tauschii and Brachypodium distachyon were clustered into one branch. HvtUF3GT was closely related to H. vulgare， while far away from Oryza sativa (indica rice) and Oryza brachyantha belonging to Trib. Oryzeae. Semiquantitative and quantitative PCR results showed that the HvtUF3GT gene was not expressed in‘INB0N7’with blue grain coat and‘Kunlun 12’ with white grain coat，while the expression level in the grain of black and purple coat was‘Heilaoya’ (black grain)>‘Kunlun 17’ (black grain)> ‘Dazhangzi’ (purple grain)> ‘Nierumuzha’ (purple grain) during the late stage of grain grouting in Tibetan hulless barley. Thus，it was speculated that the HvtUF3GT gene could play an important role in the process of grain coloration in Tibetan hulless barley.