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A group of aptamers possessing high specificity and affinity for creatine kinase MB (CKMB) was obtained by magnetic systematic evolution of ligands by exponential enrichment. Two aptamers (referred to as C.Apt.21 and C.Apt.30) were found to possess adequately low K d values. They form a well suited pair for CKMB binding. By using fluorescent microspheres, an aptamer-based lateral flow assay was developed. It is portable, economical, and sensitive. The limit of detection for CKMB is as low as 0.63 ng·mL -1 , and the assay works in the 0.005 - 2 μg·mL -1 CKMB concentration range. The method is specific for CKMB, and biomarkers for AMI (such as cardiac troponin I and myoglobin) and serum do not interfere. The strip is highly accurate as shown by analysis of spiked serum samples which gave recoveries ranging between 88 and 117%. Graphical Abstract Schematic of the test strip and sandwich aptamer-based fluorometric lateral flow assay for creatine kinease. The detection is based on the specific affinity between CKMB and selected aptamers to form a sandwich structure.