Abstract: Early diagnosis of acute myocardial infarction (AMI) is critical to saving lives. Creatine kinase isoenzyme (CK-MB) is an important biomarker for detecting AMI. A rapid detection technology was established based on near infrared fluorescence lateral chromatography. Dylight 800 with 777 and 792 nm excitation light and fluorescence was used as the fluorescence marker. The concentration of CK-MB in the sample can be accurately calculated by measuring the ratio of the peak area of the T-line to the C-line. The CK-MB test method established in this study has the characteristics of low cost, fast and accurate quantification. The results of 25 cases of clinical specimens and Beckman biochemical analysis were compared, the two detection methods have a good correlation, R²=0.98. The CK-MB test reagent established in this study can meet the rapid detection of AMI in emergency patients.