We report the chemical substitution-mediated doping of silver atoms into Au10SG10 (SG: glutathione) catenane nanoclusters (NCs) in a controllable manner. Two levels of doping were conducted leading to two silver-doped Au10–xAgxSG10 nanoclusters (with x = 0–2 and x = 1–4). Optical studies reveal that nanoclusters with a high level of doping (x = 1–4) show a blue shift in absorption and a significant red shift of ∼50 nm in the two-photon-excited emission spectrum with respect to pristine Au10SG10. The preservation of the catenane structure is confirmed by comparing the experimental and computational X-ray diffraction patterns and absorption spectra of Au10SG10 and its silver-doped analogs. In order to better address the structure–property relationship in such catenane clusters, further theoretical investigations were performed at the DFT level. A high-energy shift of the S1 state is evidenced in the calculated one-photon absorption spectra due to a larger s to d gap in silver caused by smaller relativistic effects in comparison to the gold atoms. Furthermore, the red shift in the two-photon excited emission band of the clusters is due to significantly larger calculated relaxation of the first excited state in nanoclusters with a high level of silver doping.