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Myeloperoxidase (MPO), a neutrophil-derived heme peroxidase involved in oxidative inflammatory responses, has been investigated as a biomarker for cardiovascular, hematological, and other inflammation-associated diseases. In this study, an aptamer-regulated rolling circle amplification (RCA)-CRISPR/Cas12a fluorescence assay was established for MPO detection in serum samples. Two aptamers, Apt-02 and Apt-14, were obtained with dissociation constants (K_D) of 57.76 and 56.88 nM, respectively, using MPO-depleted human serum as the counter-selection matrix during MB-SELEX. Apt-14 exhibited relatively good stability in serum and was therefore used in the assay development. Upon MPO binding, cDNA was released from the aptamer/cDNA duplex, initiating RCA and subsequently activating Cas12a-mediated trans-cleavage to amplify the fluorescence signal. Under the optimized conditions, the assay achieved a detection limit of 0.422 ng/mL with good selectivity. It also showed limited interference from the tested cardiovascular disease-related proteins and provided MPO levels in clinical serum samples that were consistent with ELISA results. The assay was also adapted to a lateral-flow strip using a biotin/FAM-labeled ssDNA reporter, enabling visual detection and reader-assisted semi-quantitative analysis.