The functionalization of gold nanoparticles with DNA has been studied extensively in solution; however, these ensemble measurements do not reveal particle-to-particle differences. Here we study the functionalization of gold nanorods with thiolated single-stranded DNA (ssDNA) at the single-particle level. We exploit the sensitivity of the plasmon resonance to the local refractive index to study the functionalization in real time using single-particle spectroscopy. We find particle-to-particle variations of the plasmon shift that are attributed to the particle size distribution and variations in ssDNA coverage. We find that the ssDNA coverage varies by ∼10% from particle to particle, beyond the expected variation due to Poisson statistics. Surprisingly, we find binding rates that differ from particle to particle by an order of magnitude, even though the buffer conditions are identical. We ascribe this heterogeneity to a distribution of activation energies caused by particle-to-particle variations in effective surface charge. These results yield insight into the kinetics of biofunctionalization at the single particle level and highlight that significant kinetic heterogeneity has to be taken into account in applications of functional particles. The presented methodology is easily extended to any nanoparticle coating and can be used to optimize functionalization protocols.