Background Isoproterenol (ISO), a synthetic on selective beta-adrenergic agonist, provides a simple and non-invasive method for inducing myocardial injury with lower mortality and higher reproducibility. Phlegm-damp syndrome, as known as "Tanshi" in Chinese, is one of Traditional Chinese Medicine (TCM) syndrome differentiation, which plays an important role in the development of cardiovascular diseases. However, the underlying mechanism remains unknown. Methods In our present study, a myocardial injury mouse model was introduced by ISO administration combined with high temperature and high humidity and high-fat diet to simulate phlegm-damp syndrome. Nontargeted metabolomics with LC-MS/MS was adopted to reveal serum metabolism profile for elucidating the possible molecular mechanism. Results The results of our study showed that phlegm-damp syndrome promoted ISO-induced myocardial injury by aggravating left ventricular hypertrophy and fibrosis, and increasing cardiac index. Our study also confirmed the presence of specific metabolites and disturbed metabolic pathways by comparing ISO mice and Tanshi mice, mainly including glycerophospholipid metabolism, arginine-proline metabolism, and sphingolipid signaling pathway. The lysoPCs, PCs, SMs, Sphingosine, and L-Arginine were the main metabolites that showed a difference between ISO and Tanshi mice, which might be the result of the underlying mechanism in the promotion of ISO-induced myocardial injury in mice with high temperature and high humidity and high-fat diet. Conclusion Our current study provides new insights into contribution of metabolism disorder in promotion of ISO-induced myocardial injury in mice with high temperature and high humidity and high-fat diet, and new targets for clinical diagnosis and pharmacologic treatment of cardiovascular disease with phlegm-damp syndrome.