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The global effort against the COVID-19 pandemic dictates that routine quantitative detection of SARS-CoV-2 neutralizing antibodies is vital for assessing immunity following periodic revaccination against new viral variants. Here, we report a dual-detection fluorescent immunochromatographic assay (DFIA), with a built-in self-calibration process, that enables rapid quantitative detection of neutralizing antibodies that block binding between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2). Thus, this assay is based on the inhibition of binding between ACE2 and the RBD of the SARS-CoV-2 spike protein by neutralizing antibodies, and the affinity of anti-human immunoglobulins for these neutralizing antibodies. Our self-calibrating DFIA shows improved precision and sensitivity with a wider dynamic linear range, due to the incorporation of a ratiometric algorithm of two-reverse linkage signals responding to an analyte. This was evident by the fact that no positive results (0/14) were observed in verified negative samples, while 22 positives were detected in 23 samples from verified convalescent plasma. A comparative analysis of the ability to detect neutralizing antibodies in 266 clinical serum samples including those from vaccine recipients, indicated that the overall percent agreement between DFIA and the commercial ELISA kit was 90.98%. Thus, the proposed DFIA provides a more reliable and accurate rapid test for detecting SARS-CoV-2 infections and vaccinations in the community. Therefore, the DFIA based strategy for detecting biomarkers, which uses a ratiometric algorithm based on affinity and inhibition reactions, may be applied to improve the performance of immunochromatographic assays. • A dual-detection fluorescent immunochromatographic assay (DFIA) was developed. • DFIA is a ratiometric detection method based on inhibition of ACE2 & RBD binding. • DFIA is self-calibrating & shows improved precision and sensitivity. • DFIA enables rapid quantitative detection of neutralizing antibodies. • Overall percent agreement between DFIA and the commercial ELISA kit was 90.98%. [ABSTRACT FROM AUTHOR]