Polygala tenuifolia root is used as a functional food due to its attractive health benefits. In this study, ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) were utilized to characterize the bioactive compounds in P. tenuifolia root. The UPLC-MS/MS information revealed 36 bioactive compounds, including oligosaccharide esters, polygalasaponins, and polygalaxanthones. GC-MS identified 34 volatile compounds with fatty acids as the main chemicals. The leading compound judged by UPLC-MS/MS was tenuifoliside A, and oleic acid was the leading volatile from GC-MS profiles. All samples tested showed similar bioactive compound compositions, but the level of each compound varied. Principal component analysis revealed the principal bioactive compounds with significant level variations between samples. These principal chemicals could be used for quality judgment of P. tenuifolia root, instead of measuring the levels of all compositional compounds.