BACKGROUND: Anxiety is highly prevalent in patients with acute myocardial infarction (AMI ), significantly impacting their quality of life. This study delved into the path mechanism of anxiety in comorbid AMI using proteomics technology, alongside behavioral and neurotransmitter assessments. The study also explored the potential myocardial protective and anxiolytic effects of LongmuShuangxinfang (LMSXF), a traditional Chinese herbal formula. METHODS: An experimental model was established by ligating the left anterior descending coronary artery and employing unpredictable empty-bottle stimulation in 18 rats divided into sham, model, and traditional Chinese medicine (TCM) groups. Proteomic analyses were conducted using label-free technology on the hippocampus and myocardial infarction marginal tissues. Parallel Reaction Monitoring (PRM) verified proteomic results and identified differential proteins. RESULTS: Proteomic and bioinformatics analyses revealed that exacerbation of anxiety symptoms in AMI might be linked to processes like the neurotransmitter receptor metabolic process, cellular response to oxidative stress, and reduced hippocampal hemoglobin levels. Myocardial injury was associated with responses to ischemia, inflammation, and calcium ion homeostasis. PRM validation highlighted the crucial roles of proteins including Gnb1, Ahsa1, Psmd14, Frrs1l, Cfl1, Aldh, and Stub1 in MI-anxiety pathogenesis. LMSXF enhanced neurotransmitter levels and anxiety status and mitigated myocardial injury by influencing hemoglobin complex levels, oxidant detoxification, neuropeptide signaling, and postsynaptic modulation.