Isolation and identification of composition from Gnaphlium affine D.Don extract were conducted with high performance liquid chromatography. Inhibition effects of extract and its three compositions on α– amylase activity were determined. The results showed that seven compositions were isolated from the extract and three of them were identified as 3,5–di–O–caffeoylquinic acid, 3,4–di–O–caffeoylquinic acid, 2′,4,4′–trihydroxy –6′–methoxychalcone–4′–O–β–D– glucopyranoside. The inhibition rates of extract, 3,5–di–O–caffeoylquinic acid, 3,4–di–O–caffeoylquinic acid, and 2′,4,4′–trihydroxy –6′–methoxychalcone–4′–O–β–D–glucopyranoside were 32.37%，84.53%, 63.07% and 71.22% respectively when the concentration of α– amylase was 1.67 mg/mL, and half inhibitory concentrations (IC50) were 2.71, 0.90, 1.28 and 1.16 mg/mL. The kinetic analysis showed that the inhibition of 2′,4,4′– trihydroxy –6′–methoxychalcone –4′–O–β–D–glucopyranoside on α–amylase was reversible and non–competitive.