Excited-state intramolecular proton transfer (ESIPT)-exhibiting polymer nanoparticles (e-PNPs) were prepared using 2-hydroxy-5-methylisophthalaldehyde and melamine as reactants. These e-PNPs possess abundant aromatic o-OH, CN and NH2 groups, which are responsible for ESIPT modules and molecular recognition. Compared with small ESIPT compounds, e-PNPs displayed ultrabright luminescence and high fluorescence quantum yield owing to their rich ESIPT-chromophores and appropriate hydrophobic environment for ESIPT reaction. The e-PNPs can emit strong green fluorescence and exhibit typical dual emission in a HEPES buffer (20 mM, pH 7.5). Upon the addition of VB1, the ESIPT reaction of e-PNPs was inhibited, eliciting obvious decrease in ESIPT fluorescence. By making use of this signaling mechanism, a tautomeric fluorescence quenching-based method for detecting VB1 was established. A good linear range of 0.1–25 μM.L−1 was observed, bringing about an excellent detection limit of 2.6 nM.L−1. Moreover, this assay shows good selectivity over other water-soluble vitamins and some biologically related species. The employment of e-PNPs for the determination of VB1 in kiwi and grape juice samples showed remarkable abilities in terms of the simplicity, stability, reproducibility and sensitivity, which conforms its great potential for real sample analysis. Therefore, e-PNPs can offer a simple, highly sensitive and selective sensing model for fluorescence detection of VB1.