To isolate the chemical constituents of ethyl acetate extract from Viola biflora. Isolation and purification were carried out on repeated silica gel column chromatography,PTLC,and Sephadex LH-20. The structures of these compounds were elucidated by physico-chemical properties and spectral analyses. Twelve compounds were isolated from Viola biflora,which identified as aurantiamide acetate( 1),solalyratin B( 2),esculetin( 3),scopoletin( 4),lupeol( 5),132S-hydroxypheophytin a( 6),vomifoliol( 7),dibutyl phthalate( 8),(-)-dihydrovomifoliol( 9),grasshopper ketone( 10),crassifol( 11) and β-sitosterol( 12). All the compounds are isolated from Viola biflora for the first time. Compounds 2,7,9 ~ 11 are isolated from Viola genus for the first time.