The flavonoids contained in Euphorbia neriifolia leaves were extracted, identified and characterized. Direct and sequential soxhlet extraction and its concentrated fractions were subjected to thin layer chromatography and high performance thin layer chromatography. The results showed that maximum yield of the flavonoid (6.53 g) was obtained from ethanolic extract. The Rf value of isolated flavonoid and phytochemical screening has been compared with standard Quercetin. Characterization of isolated flavonoid was done by IR, 1 H NMR, and MS. On the basis of chemical and spectral analysis structure was elucidated as 2-(3,4-dihydroxy-5-methoxy-phenyl)- 3,5-dihydroxy-6,7-dimethoxychromen-4-one, a flavonoid. This compound was isolated for the first time from this plan