<p> An accurate method is presented for quantifying recombinant neuron-specific enolase (NSE) using isotope dilution liquid chromatography–mass spectrometry (IDMS) based on amino acid analysis. Traceable to the International System of Units − moles, the NSE concentration was determined as (0.0924 ± 0.0074) mg∙mL^− 1. The quantified NSE was then applied as a calibrator in a magnetic nanoparticle-based immunochromatographic assay (MNP-ICA) for point-of-care detection of NSE in human serum. Under the optimized parameters, the MNP-ICA exhibited a linear range from 2.5 ng∙mL^− 1 to 80.0 ng∙mL^− 1, with a detection limit of 2.09 ng∙mL^− 1. Intra- and inter-assay precision showed relative standard deviations below 4.5% and 11.6%, respectively, and recoveries ranged between 88.8% and 107.3%. Results from 16 actual human serum samples correlated well with a commercial ELISA kit.&#xa0;This agreement between the two immunoassays underscores the reliability of the MNP-ICA and confirms the immunoreactivity of the quantified NSE in clinical samples.</p> Graphical Abstract <p></p>