Glycyrrhiza uralensis Fisch. (G. uralensis) is one of the most widely used herbal medicines. This study was designed to enrich total flavonoids (TFF) from G. uralensis. The chemical profile of TFF was identified by HPLC and colorimetric assay. The TFF mainly contained liquiritin apioside, liquiritin, isoliquiritin apioside, liquiritigenin and isoliquiritigenin without glycyrrhizic acid. To study the anti-inflammatory activity of TFF, the DMB-induced ear vasodilatation assay and carrageenan-induced rat paw edema model have been utilized. Treatment with TFF showed significant anti-inflammatory activities in the two models. The two in-vivo edema assays demonstrated that the TFF possesses significant dose-dependent anti-inflammatory activity, similar to that of indomethacin at a dose of 500 mg/kg. In rat paws with carrageenan, treatment with TFF (500 and 250 mg/kg) markedly inhibited the expression of IL-1β and iNOS. TFF at all doses noticeably decreased levels of NO and MDA at the site of inflammation, while only i.g. TFF at a dose of 500 mg/kg significantly decreased TNF-α levels in the carrageenan-injected paws. In addition, an increase in SOD activity was induced by TFF at all doses. These results revealed that TFF exhibited significant anti-inflammatory activity in acute inflammatory models.