Background: Diabetes mellitus is a disease caused by a malfunction in the way carbohydrates are metabolized. It is frequently associated with abnormal insulin levels in the blood and organ insulin resistance. Aim: Antidiabetic activity of Flaveria trinervia ethanolic extract. Objective: Phytochemical analysis, In-vitro, and In-vivo activity antidiabetic activity of F. trinervia ethanolic extract. Material and methods: F. trinervia were collected from Kovilpatti village, Thoothukudi district, Tamil Nadu, India in June 2021. The air-dried whole plant was crushed, and 500 gm of the powder, ethanol was used for continuous extraction by the Soxhlet device. Extract phytochemical analysis done by conventional and GC-MS analysis. The antidiabetic activity of F. trinervia ethanolic extract was investigated in Wistar albino rats. Results: F. trinervia ethanolic extract were prepared by using the Soxhlet system. F. trinervia ethanolic extract showed α-amylase inhibitory activity with IC50 value 125 µg/mL when compared with the standard acarbose. F. trinervia ethanolic extract conducted glucose uptake assay in 3T3-L1 cell lines and showed efficient glucose uptake enhancement capability. In-vivo antidiabetic studies confirmed that F. trinervia ethanolic extract has good antidiabetic properties. F. trinervia phytoconstituents are responsible for antidiabetic activity. Conclusion: The study suggested treating antidiabetic disease can be used in F. trinervia ethanolic extract. Further in detailed study need to understand the mechanism of action of antidiabetic activity