Heat shock transcription factor (Hsf) exists widely in eukaryotes and responds to various abiotic stresses by regulating the expression of downstream transcription factors, functional enzymes and molecular chaperones. In this study, the seedlings of wheat (Triticum aestivum)‘Cang 6005’ was used as material. The target gene ^TaHsfA25 was isolated from young leaves treated by heat shock at 37 ℃ using homologous cloning method, the subcellular location of the gene was analyzed, and the wild type Arabidopsis thaliana plants were transformed by agrobacterium tumefaciens mediated dipflower method. The positive seedlings were screened for basal and acquired thermotolerance analysis, and the expression of HSR gene of transgenic Arabidopsis under heat stress was analyzed for exploring the biological function of ^TaHsfA25. The results show that: (1) the wheat heat shock transcription factor ^TaHsfA25 was successfully obtained, which encoded 405 amino acids with a molecular weight of 44.9 kDa. Sequence alignment showed that TaHsfA25 protein of wheat had the highest similarity with HvHsfA2b protein of barley, which reached 88.07%. (2) The observation results of laser confocal microscope showed that TaHsfA25 was located in the nucleus. (3) qRTPCR analysis showed that ^TaHsfA25 was highly expressed in mature roots, stamens, pistils, sepals, mature and immature embryos of wheat, and under heat stress, ^TaHsfA25 showed different upregulated expression trends in wheat leaves and roots. (4) Phenotypic observation showed that transgenic Arabidopsis with ^TaHsfA25 could improve the basal and acquired thermotolerance of seedlings under heat stress when compared with wildtype, while restored the thermotolerance phenotype of the deletion mutant AtHsfa2. The chlorophyll content of different lines after heat stress was consistent with the phenotypes. (5) HSR gene expression analysis showed that overexpression of ^TaHsfA25 in Arabidopsis upregulated HSR gene expression levels to varying degrees, suggesting that ^TaHsfA25 may enhance thermotolerance by regulating the expression of multiple heat stress related genes in Arabidopsis.