Despite the rapid development of nanomaterials and nanotechnology, it is still desirable to develop novel nanoparticle-based techniques which are cost-effective, timesaving, and environment-friendly, and with ease of operation and procedural simplicity, for assay of target analytes. In the work discussed in this paper, the dye fluorescein isothiocyanate (FITC) was conjugated to 1,6-hexanediamine (HDA)-capped iron oxide magnetic nanoparticles (FITC-HDA FeO MNPs), and the product was characterized. HDA ligands on the surface of FeO MNPs can bind 2,4,6-trinitrotoluene (TNT) to form TNT anions by acid-base pairing interaction. Formation of TNT anions, and captured TNT substantially affect the emission of FITC on the surface of the FeO MNPs, resulting in quenching of the fluorescence at 519 nm. A novel FITC-HDA FeO MNPs-based probe featuring chemosensing and magnetic separation has therefore been constructed. i.e. FITC-HDA FeO MNPs had a highly selective fluorescence response and enabled magnetic separation of TNT from other nitroaromatic compounds by quenching of the emission of FITC and capture of TNT in aqueous solution. Very good linearity was observed for TNT concentrations in the range 0.05-1.5 μmol L, with a detection limit of 37.2 nmol L and RSD of 4.7 % ( n = 7). Approximately 12 % of the total amount of TNT was captured. The proposed methods are well-suited to trace detection and capture of TNT in aqueous solution. [Figure not available: see fulltext.]