The unclear composition of bitter substances in peony seeds limits the comprehensive development and efficient utilization of peony seeds. In this study， a combination of organic solvent extraction and sensory evaluation was used to separate and identify the bitter substances in peony seeds by ultra-high performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry (UPLC-HRMS). The results of the study showed that the peony seed sample was extracted by n-hexane， citric acid aqueous solution， n-butanol， and purified by AB-8 macroporous resin. 5 components were obtained: F-Ⅱ-1-MR1， F-Ⅱ-1-MR2， F-Ⅱ-1-MR3， F-Ⅱ-1-MR4， F-Ⅱ-1-MR5. Two bitter components were screened by sensory evaluation: F-Ⅱ-1-MR2， F-Ⅱ-1-MR3. UPLC-HRMS was used to analyze and identify those bitter components. A total of 24 compounds were identified， including 7 monoterpene glycosides， such as paeoniflorin， 7 flavonoids， such as luteolin， 2 stilbene， such as resveratrol， 6 fatty acids， such as 9，12，13-trihydroxy-10，15-octadecadienoic acid， and 1 kind of organic acids was benzoic acid. Among them， paeoniflorin is the highest yield component in peony seed extract， and it is also the main bitter compound in peony seeds.