Thiolate coordination to zinc(ii) ions occurs widely in such functional biomolecules as zinc enzymes or zinc finger proteins. Here, we introduce a simple method for determining the affinity of ligands toward the zinc-enzyme active-center model tetramethylrhodamine (TAMRA)-labeled 1,4,7,10-tetraazacyclododecane (cyclen)-zinc(ii) complex (TAMRA-ZnL). The 1 : 1 complexation of TAMRA-labeled cysteine (TAMRA-Cys) with TAMRA-ZnL (each at 2.5 μM), in which the TAMRA moieties approach one another closely, induces remarkable changes in the visible absorption and fluorescence spectra at pH 7.4 and 25 °C. The 1 : 1 complex formation constant (K = [thiolate-bound zinc(ii) complex]/[uncomplexed TAMRA-ZnL][uncomplexed TAMRA-Cys], M-1) was determined to be 106.7 M-1 from a Job's plot of the absorbances at 552 nm. By a ligand-competition method with the 1 : 1 complexation equilibrium, analogous K values for thiol-containing ligands, such as N-acetyl-l-cysteine, l-glutathione, and N-acetyl-l-cysteinamide, were evaluated to have similar values of about 104 M-1. As a result of the ligand affinities to TAMRA-ZnL, nonlabeled zinc(ii)-cyclen induced remarkable stabilization of the reduced form of l-glutathione and a cysteine-containing enolase peptide to aerial oxidation in aqueous solution at pH 7.4 and 25 °C.