AGO protein is the core molecule of the RNAinducible silencing complex, which plays an important role in plant growth, development and stress response. In order to explore the mechanism of AGO gene in Tibetan hulless barley resistance to barley leaf stripe, we obtained a differentially expressed gene from the transcriptional sequencing results before and after infection with barley leaf stripe in resistant Tibetan hulless barley variety ‘Kunlun14’ and the susceptible variety ‘Z1141’. The results showed that: (1) HvtAGO1 was isolated from leaves of ‘Kunlun 14’ and ‘Z1141’. We obtained a 3 675 bp sequence which contained a 3 651 bp open reading frame (ORF), encoding 1 217 amino acids in this study. Protein sequence analysis showed that HvtAGO1 was a hydrophilic unstable basic protein with highly conserved DUF1785, PAZ and PIWI domains. (2) The sequence similarity of the AOG1 protein between Tibetan hulless barely and Aegilops tauschii, Brachypodium distachyon and Triticum urartu was 96.64%, 79.34 and 80.07%, respectively, and the domain similarity of DUF1785, PAZ and PIWI was 97.57%, 98.44% and 96.02%, respectively. Phylogenetic analysis showed that the affinity of HvtAGO1 protein of Tibetan hulless barley was closest to that of A. tauschii, but far from that of Zea mays and Setaria italica. (3) The degradation sequencing proved that HvtAGO1 was the target gene of hvumiR1685p. (4) qRTPCR results showed that the expression levels of hvumiR1685p and the target gene HvtAGO1 were significantly upregulated in ‘Kunlun 14’ and ‘Z1141’ compaired with normal leaves, respectively. Meanwhile, the gene expression levels of ‘Kunlun 14’ were significantly higher than that of ‘Z1141’ under barley leaf stripe. Research speculated that hvumiR1685p and target gene HvAtGO1 play an important role in the regulation of resistance to barely leaf stripe in Tibetan hulless barley.