Precisely regulating the ligand exchange on the nanocluster (NC) surface is challenging but important for fully understanding metal-ligand interactions and transformations. In this work, the homosilver nanocluster Ag40(TBBM)22(CH3COO)10 (Ag40) and its Au-substitution derivative AuAg39(TBBM)20(CH3COO)12 (AuAg39) have been synthesized and characterized by single-crystal X-ray crystallography (SC-XRD), nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), and thermogravimetric analysis (TGA). By precisely controlling the template metal exchange progress, the intermediate product, namely, AuAg39(TBBM)21(CH3COO)11 (AuAg39-inter), was also obtained and then characterized by SC-XRD. These three nanoclusters possess a similar metallic framework, albeit some of the thiol ligands of Ag40 have been replaced by the introduced acetate ligands in both AuAg39-inter and AuAg39. Furthermore, Ag40 and AuAg39 NCs can serve as chiral amplifiers for testing the ee values of 2-chloropropionic acid, ibuprofen, naproxen, and isoleucine with high sensitivity.