Moutan Cortex, is the root bark of Paeonia suffruticosa Andrews, which is classified into three specifications according to whether or not it is peeled and cored: Liandanpi, Guadanpi and whole root. In this study, the cork layer, cortex, phloem and xylem of P. suffruticosa fresh root were precisely separated by laser microdissection technique. UPLC-Q-Orbitrap-MS and UPLC-QQQ-MS techniques were used to analyse the differences in the chemical composition of different tissue parts of P. suffruticosa fresh root and Liandanpi, and to determine the optimal processing method of P. suffruticosa root. As a result, a total of 90 compounds were characterised, among which the cork layer had more types and higher contents of chemical constituents, and the xylem had fewer types and lower contents of chemical constituents. The proportion of xylem is larger, while the type and content of active ingredients is smaller. Therefore, the processing method of removing the wood core and retaining the cork bark can be used in the processing of Moutan Cortex. In this study, laser microdissection and ultra performance liquid chromatography-mass spectrometry were used to provide a theoretical basis for optimising the processing method of Moutan Cortex to enhance its pharmacological effects.