Myocardial infarction (MI) is the leading cause of death worldwide. Here, we present a novel, label-free biosensor for detecting myocardial ischemia biomarkers via CRISPR/Cas12a. This system utilizes the unique properties of CRISPR/Cas12a and G-quadruplex-ThT-based biosensors, enabling sensitive and specific detection of ATP, a crucial biomarker in cardiovascular diseases, at concentrations as low as 23 nM. Our method demonstrates substantial improvements over traditional ATP detection techniques, such as high-performance liquid chromatography and enzymatic assays, which often require complex sample preparation methods and costly equipment. The feasibility of the biosensor was further demonstrated in various models, including heart failure in mice and hypoxic conditions in cardiomyocytes. This successfully showcased its ability to function as a practical tool for diagnosing and monitoring diseases characterized by ATP dysregulation, highlighting its effectiveness in real-world clinical scenarios. This biosensor is notable for its rapid response, ease of use, and potential for integration into point-of-care diagnostics. These features offer significant advantages for the early diagnosis and management of ischemic heart disease and other conditions where ATP serves as a key metabolic biomarker. This technology also offers significant potential for early diagnosis and monitoring of myocardial ischemia and cardiovascular diagnostics. These findings underscore the biosensor's capacity for real-time ATP monitoring, offering crucial insights into mitochondrial function and disease progression, particularly in cardiovascular and inflammatory diseases.