Human cells generate a bulk of aldehydes during lipid peroxidation (LPO), influencing critical cellular processes, such as oxidative stress, protein modification, and DNA damage. Enals, highly reactive α,β-unsaturated aldehydic metabolites, are implicated in various human pathologies, especially neurodegenerative disorders, cancer, and cardiovascular diseases. Despite their importance, endogenous enals remain poorly characterized, primarily due to their instability and low abundance. Herein, we introduced "enalomics," a mass spectrometry (MS)-based approach for profiling, identifying, and semiquantifying enals in biological samples. Derivatization with 2,4-dinitrophenylhydrazine and treatment with ascorbic acid stabilized enals in biological matrices and provided a unique MS fragment ([M-H-47]-) for reliable enal identification. Utilizing precursor ion scanning, dynamic multiple reaction monitoring, high-resolution MS, and mathematical correlations between retention times and carbon numbers of enals, we identified 157 enals (127 newly reported) with tissue-specific profiles in rats and 29 enals (24 newly reported) in human plasma. To the best of our knowledge, this represents the comprehensive analysis of enals, i.e., "enalomics," in biological samples. Enalomics demonstrated significant alterations in enal metabolism in rats with myocardial injury, highlighting the potential of medium- and short-chain plasma enals as sensitive diagnostic biomarkers. Further application of enalomics in patients with myocardial infarction (MI) identified 14 plasma diagnostic biomarkers. Receiver operating characteristic curves showed good discrimination (area under curve ≥ 0.8603, p ≤ 0.0043). This research advances the understanding of LPO products and emphasizes the roles of enals in human diseases, offering good prospects for early screening, diagnosis, and clinical interventions targeting LPO products in MI patients.