Enzyme-mimicking nanoparticles play a key role in important catalytic processes, from biosensing to energy conversion. Therefore, understanding and tuning their performance is crucial for making further progress in biological applications. We developed an efficient and sensitive electrochemical method for the real-time monitoring of the glucose oxidase (GOD)-like activity of single nanoparticle through collision events. Using brush-like sulfonate (-SO3-)-doped polyaniline (PANI) decorated on TiO2 nanotube arrays (TiNTs-SPANI) as the electrode, we fabricated a proton reservoir with excellent response and high proton-storage capacity for evaluating the oxidase-like activity of individual Au nanoparticles (AuNPs) via instantaneous collision processes. Using glucose electrocatalysis as a model reaction system, the GOD-like activity of individual AuNPs could be directly monitored via electrochemical tests through the nanoparticle collision-induced proton generation. Furthermore, based on the perturbation of the electrical double layer of SPANI induced by proton injection, we investigated the relationship between the measured GOD-like activities of the plasmonic nanoparticles (NPs) and the localized surface plasmon resonance (LSPR) as well as the environment temperature. This work introduces an efficient platform for understanding and characterizing the catalytic activities of nanozymes at the single-nanoparticle level.