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A one-pot, simple and convenience method to synthesize Fe3O4 nanocrystal clusters.Highly sensitive and magnetic quantitative lateral flow immunoassay is developed.Trace-level antibody, NT-proBNP, is detected at a point-of-care manner.Quantitative linear relationship between signal and antibody concentration is set up.Lateral flow immunoassay (LFIAs) has found widespread applications in point-of-care testing (POCT). However, conventional LFIAs based on gold nanoparticles can only provide limited information (qualitative or semi-quantitative information) and suffer from low sensitivity. To address this issue, we developed a Fe3O4 nanocrystal cluster-based highly sensitive LFIAs for the simultaneously qualitative and quantitative detection of trace-level biomarkers. The synthesis of poly (acrylic acid)-stabilized superparamagnetic nanocrystal clusters (PAA-MNCs) is a one-pot, simple and convenient technique, and the polyelectrolyte coating provides enough stability, good dispersity and downstream conjugation sites. Using these PAA-MNCs as a label nanomaterial in LFIAs, a model biomarker (amino-terminal pro-B-type natriuretic peptide (NT-proBNP) antibody) was detected qualitatively and quantitatively. The results show that tests have a qualitative detection limit of 100 pg/mL and a quantitative linear relationship between the magnetic signal and antibody concentration (20–8000 pg/mL) under the optimized experimental conditions. The developed LFIA based on PAA-MNCs for NT-proBNP could be used for the clinical assessment of heart failure, offering tremendous potential for personalized medicine.