Migao volatile oil (MVO), a traditional Chinese medicine extract, exerts a curative effect on coronary heart disease (CHD). In this study, gas chromatography-mass spectrometry (GC-MS) was used to determine the fingerprints of 10 MVO batches, and 27 common components were identified. The anti-CHD activities of the different MVOs were determined using ELISA kits, and haematoxylin and eosin staining was used to assess the histopathological injury in myocardial tissues. Three mathematical models were used to analyse multiple bioactive indices, and an anti-CHD-guided spectrum-effect relationship approach was developed to discover MVO quality control (QC) markers. α-phellandrene, p-cymene, 1,8-cineole, linalool, α-terpineol, α-cubebene, α-copaene, α-humulene, dihydroagarofuran, and δ-cadinene all showed good affinity for target proteins connected to CHD, showing their potential as QC markers. Based on the anti-CHD-guided spectrum-effect relationship approach, a bioactive constituent-based holistic QC method was successfully developed for MVO, which could provide a valuable reference for related QC research.