By combining single-molecule fluorescence microscopy with traditional electrochemical methods, herein we report on the investigation of the electrocatalytic kinetics of two-electron (2e) pathway of oxygen reduction reaction (ORR) on a single Fe3O4 nanoparticle. The kinetic parameters for two-electron ORR process are successfully derived at the single-particle level, and a potential dependence of dynamic heterogeneity among individual nanoparticles is revealed. Furthermore, the performance stability of individual Fe3O4 nanoparticles for 2e ORR process is studied. This study deepens our understanding to the electrocatalytic ORR process, especially the 2e pathway at single-molecule and single-particle levels.