In this study we analysed the expression patterns of TaLr35PR2 and confirmed its role in Lr35-mediated adult resistance to leaf rust fungus. β-1,3-glucanase, a pathogenesis-related protein, has a critical function in plant defence response against fungal pathogens. We previously described the full-length gene TaLr35PR2, which encodes a protein exhibiting amino acid and structural similarity to β-1,3-glucanase, in the wheat near-isogenic line TcLr35 (GenBank accession number DQ294235.1). This work aimed to further assess TaLr35PR2 expression patterns and function in Lr35-mediated adult resistance to Puccinia triticina. Immunoblot was performed to demonstrate that TaLr35PR2 expression was triggered early by P. triticina, with expression levels markedly elevated in incompatible interaction compared with those in compatible one. Additionally, TaLr35PR2 accumulation steadily increased and overtly peaked after challenge with P. triticina through the various developmental stages of TcLr35 wheat, and remaining at similar levels after mock inoculation. Furthermore, TaLr35PR2 expression was significantly reduced in barley stripe mosaic virus (BSMV)-induced gene knockdown plants, in which pathological assessment revealed that TaLr35PR2-silenced plants was obviously susceptible to leaf rust fungus compared with wild-type TcLr35, indicating that Lr35-mediated resistance to leaf rust was diminished. These findings strongly suggest that TaLr35PR2 is involved in Lr35-mediated wheat defence against the leaf rust pathogen.