Abstract: 【Objective】 To explore the function of the promoter of the flavanone-3-hydroxylase gene in pansy（Viola×wittrockiana Gams）（VwF3H） and verify its interaction with anthocyanin-related MYB transcription factors, which could provide reference for revealing the regulatory mechanism of F3H gene in the process of plant anthocyanin synthesis. 【Method】Using pansy as the research materials, the VwF3H gene promoter sequence was cloned by FPNI-PCR, and its functional elements, tissue expression specificity and core functional fragments were analyzed. Yeast one-hybrid assay was conducted to investigate whether VwMYB27 and VwMYB29 could bind to the VwF3H gene promoter. 【Result】 VwF3H gene promoter sequence was 2017 bp long and contained numerous light-responsive and hormone-responsive elements. Based on the positions of important transcription factors such as MYB, it was divided into 4 segments: VwF3H1（2017 bp）, VwF3H2（1353 bp）, VwF3H3（745 bp） and VwF3H4（446 bp）. The relative expression of VwF3H gene was the highest in pansy petals, significantly higher than in roots, stems and leaves（P＜0.05, the same below）. The fulllength VwF3H gene promoter exhibited strong transcriptional activity. The transcriptional activity of VwF3HS2, with a deletion from-2017 to-1353 bp, was greatly reduced, while VwF3HS3 and VwF3HS4, with more bases deleted, showed almost no transcriptional activity, indicating that the high-activity sequence of the VwF3H gene promoter was located in the-2017 to-1353 bp region. The ability of the VwF3H gene promoter to drive GUS gene expression differed between tobacco leaves and flowers, with stronger transcriptional activity in floral organs, suggesting tissue-specific expression of VwF3H gene. Additionally, the transcriptional activity of VwF3H gene promoter in tobacco was significantly lower than that of the CaMV35S promoter, but it was significantly higher than that of the CaMV35S promoter in pansy, suggesting species preference of the VwF3H gene promoter and its suitability as a promoter for petal transformation vectors in pansy. The self-activation inhibition concentration of 3-amino-1,2,4-triazole（3-AT） for VwF3H gene promoter was 250 mmol/L. VwMYB27 interacted with VwF3H gene promoter, whereas VwMYB29 did not directly interact with it. 【Conclusion】 VwF3H gene promoter exhibits strong transcriptional activity in pansy, with its high-activity sequence located in the-2017 to-1353 bp region. VwF3H gene promoter interacts with VwMYB27 and is involved in the formation of petal color patterns in pansy.