A novel choline detection strategy is developed based on in situ polymerization of aniline on the surface of upconverting nanoparticles (UCNPs). In acidic buffer solution, aniline was protonated and attached to the surface of negatively charged UCNPs via electrostatic interactions. The in situ polymerization of aniline was initiated with the addition of a catalyst (HRP) and an oxidization agent (H2O2). The upconversion luminescence was efficiently quenched by polyaniline (PANI), and the quenching efficiency could reach 97.5%, which was higher than most of the reported organic quenchers for UCNPs. In the presence of choline oxidase, choline was hydrolyzed and produced H2O2, which caused production of polyaniline and quenching of upconversion luminescence. A sensing method for choline is thus developed. The upconversion luminescence based choline detection method can very much avoid the interference of fluorescent substances in biological samples, and also possesses the advantages of good selectivity, sensitivity and convenience.