Metal nanoparticles, clusters, and single atoms exhibit remarkable variations in catalytic performance due to their different electronic and atomic structures. To explore the size-dependent effects on acetylene hydrochlorination, a series of Ru catalysts (including single atoms (Ru SAC CS), clusters (Ru ACs CS), and nanoparticles (Ru NPs CS) catalysts) were accurately synthesized by a defect-engineering strategy. Ru SAC CS demonstrated the optimal catalytic performance. The structural–activity relationship between the catalyst’s initial activity and charge, Ru–Ru coordination number and the oxidation state of Ru sites offer insights into how the structure of Ru active sites affects acetylene hydrochlorination at the atomic scale. Density functional theory (DFT) simulations reveal that the energy barrier for the rate-determine-step (*Cl approaching the *CH₂=CH intermediate to form *C₂H₃Cl) for Ru SAC CS is significantly lower, facilitating barrier overcoming and enhancing vinyl chloride formation. Furthermore, Ru SAC CS displays suitable adsorption energies for C₂H₂ and C₂H₃Cl, which is conducive to prevent coke deposition and enhance the catalytic stability. This research demonstrates the efficiency of Ru single-atom catalysts for acetylene hydrochlorination and offers new perspectives on the precise construction and catalytic mechanism of sub-nanometer catalysts.