AbstractQuantification of nanoparticle-molecule interaction at a single-molecule level remains a daunting challenge, mainly due to ultra-weak emission from single molecules and the perturbation of the local environment. Here we report the rational design of an intraparticle-surface energy transfer (i-SET) process, analogous to high doping concentration-induced surface quenching effects, to realize single-molecule sensing by nanoparticle probes. This design, based on a Tb³⁺-activator-rich core-shell upconversion nanoparticle, enables a much-improved spectral response to fluorescent molecules at single-molecule levels through enhanced non-radiative energy transfer with a rate over an order of magnitude faster than conventional counterparts. We demonstrate a quantitative analysis of spectral changes of one to four fluorophores tethered on a single nanoparticle through i-SET spectroscopy. Our results provide opportunities to identify photoreaction kinetics at single-molecule levels and provide direct information for understanding behaviors of individual molecules with unprecedented sensitivity.