BACKGROUND To establish a fast and simple quantitative method for detection of heart-type fatty acid-binding protein (H-FABP) in serum based on a background fluorescence quenching immunochromatographic assay. METHODS A detection card based on the double-antibody sandwich double-antibody method with background fluorescence quenching was developed for quantitative measurement of H-FABP in serum. The optimal concentrations of control for coating the test and control lines were determined as well as the concentrations of gold-labeled antibodies used in preparing the detection system. The detection method for H-FABP in serum was established and validated using real-world clinical samples. RESULTS The optimal concentrations of labeling antibody and coating antibody were 5.0 μg/mL and 1.0 mg/mL, respectively. The test card had a sensitivity of 1.15 ng/mL over a linear concentration range of 0-100 ng/mL. Based on three batches prepared for testing the card, the relative standard deviation (RSD) within batches was less than 15% without a significant difference (P=0.942). The detection method was tested against common interfering substances in serum, such as bilirubin, triglyceride and serum anticoagulants ethylenediamine tetraacetic acid (EDTA), heparin, and sodium citrate, and no significant cross-reaction was detected. The test method was further validated with 50 clinical serum samples, and the test results were comparable with standard reference detection methods with good correlation (R=0.95). CONCLUSIONS Our study presents a new method with strong specificity and sensitivity for the detection of H-FABP in serum, which could promote H-FABP detection in a broad range of applications.