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This paper reports a facile synthesis of bimetallic Au x Pt y nanozyme using C-rich oligonucleotide as the nucleation template, with high peroxidase-like activity and thiophilicity associated with the ratio of x : y . The efficient Au 2 Pt 1 nanozyme possesses highly peroxidase mimicking activity with the K m value of 0.088 mM toward 3,3′,5,5′-tetramethylbenzidine and 196 mM toward hydrogen peroxide. It is the first report to explore colorimetric assays for biothiols with high sensitivity and selectivity against non-thiol-containing amino acids, on the basis of thiol-induced inhibition of peroxidase-like activities of metal nanoparticles. By using Au 2 Pt 1 nanozyme, the limit of detection (LOD) is 3.5 nM for cysteine and 1.6 nM for homocysteine. This DNA-stabilized bimetallic Au x Pt y nanozyme shows a promising application in quantitative detection of biothiols in biological fluids. [ABSTRACT FROM AUTHOR]