Mast cell tumors (MCTs) are the most common skin tumors in dogs. Clinical data and cytological examination typically suffice for the initial diagnosis. However, prognosis and appropriate treatments necessitate histopathological examination. Amid global concerns regarding hazardous chemicals, the utilization of natural dyes has garnered increased attention. In this context, we focused on using butterfly pea flower (BPF) extract, rich in anthocyanins, as a dye for canine histopathological MCT staining. The dried petal powder was dissolved in distilled water (DW), filtered to obtain a crude extract, and then subjected to a freeze-drying process to preserve its quality. The BPF-dye was prepared by adding DW and 2.5% NaCl into the dried extract. The paraffin serial sections of 32 diagnosed MCTs were stained with the dye for 2 hours. Additionally, the sections were stained with Hematoxylin and Eosin, and Toluidine blue (TB) routine dyes for MCT diagnosis. Sections of non-MCT round cell tumors were also stained with BPF-dye. The results revealed pink-reddish specific staining in the neoplastic mast cell granules, with different staining levels corresponding to the degree of cytoplasmic granulation, similar to TB staining. However, TB staining exhibited blue or metachromasia, indicating differences in staining color. Additionally, the BPF-dye could differentiate MCT from non-MCT round-cell tumors, providing an alternative method of differentiation. The dye maintained its staining ability for 60 days when stored at 4^oC. Accordingly, it holds potential as an alternative dye for MCT diagnosis, being locally available and eco-friendly. Further study should focus on improving the stability and longevity of the dye by preserving its anthocyanin content with natural co-pigments for practical application.