A Pt-NiCo nanomaterial has been synthesized for developing the sensitive electrochemical determination of biological thiols that include L-cysteine (CySH), homocysteine (HCySH), and gluthione (GSH) with high sensitivity and long-term stability, in which the Pt nanoparticles are well supported on amorphous NiCo nanofilms. The electrochemical oxidation of thiols has been successfully facilitated on the optimized Pt-NiCo nanostructures, that is, two oxidation peaks of CySH have been clearly observed at potentials of +0.06 and +0.45 V. The experimental results demonstrate that the first peak for CySH oxidation may be attributed to a direct oxidation from CySH to L-cystine (CySSCy), whereas the second peak possibly results from a sequential oxidation from CySSCy to cysteic acid (CySO(3)H), together with a direct oxidation of CySH into CySO(3)H. The enhanced electrocatalytic activities at the Pt(23)-NiCo nanostructures have provided a methodology to determine thiols at a very low potential of 0.0 V with relatively high sensitivity (637 nA μM cm(-2)), a low detection limit (20 nM), and a broad linear range. The striking analytical performance, together with the characteristic properties of the Pt-NiCo nanomaterial itself, including long-term stability and strong antipoisoning ability, has established a reliable and durable approach for the detection of thiols in liver cancer cells, Hep G2.