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A reliable, rapid analytical method was established for characterization of constituents in the ethanol extract of Polygonum multiflorum by combining an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). 131 constituents which including phenolic acids, stilbenes, flavones, anthraquinones, naphthalenes and their derivatives were identified or tentatively identified by using characteristic diagnostic fragment ions and references. The established method was further applied to analyze blood samples, and successfully identified 41 compounds which were absorbed through the gastrointestine in rats after administration the extract of P. multiflorum. Moreover, the pharmacokinetic studies of some major compounds in blood were investigated by using ultra performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) method. This study showed a comprehensive research of P. multiflorum, which could provide a meaningful basis for further quality control, pharmacological as well as toxicological researches.A sensitive UHPLC-Q-TOF/MS method was established to identify 131 compounds in the ethanol extract of Polygonum multiflorum.41 compounds which were absorbed through gastrointestine in rats after administration the extract of P. multiflorum were identified.Seven potential bio-marker compounds in PM extract were quantitative analyzed using UPLC–MS/MS method.The pharmacokinetic behaviors of the 7 compounds in rats were investigated by using sensitive UPLC–MS/MS method.