l-cysteine (Cys) is an important biomolecule responsible for many biological processes including gene regulation and intracellular signaling. Therefore the detection of Cys in biological samples is highly needed in the recent years. In the present study, we describe the use of gold/silver alloy nanoparticles (Au/Ag alloy NPs) as a probe for the colorimetric detection of Cys using UV-vis spectrophotometric method with high sensitivity and selectivity. Au/Ag alloy NPs were prepared by chemical reduction method and their morphological properties were characterized by using various analytical techniques. After the addition of various Cys concentrations into Au/Ag alloy NPs, the surface plasmon resonance (SPR) band at 410 nm decreases continuously and simultaneously a new peak appears at 624 nm, which confirmed the agglomeration of alloy NPs. In addition to that, the aggregation process of Au/Ag alloy NPs was evident from the change of color from yellow to colorless by naked eye. The results clearly demonstrated that the formation of aggregated structure of Au/Ag alloy NPs-Cys and also the formation of Ag-S bond and electrostatic interaction between alloy NPs and Cys molecules. Additionally, the Au/Ag alloy NPs showed good selectivity for Cys detection even in the presence of other amino acids. Furthermore, the current approach showed good linearity in 2.00 mu M to 18.00 mu M range, with a limit of detection of 0.42 mu M and a correlation value of 0.9934. The present technique was successfully utilized for the detection of Cys in human serum samples. As a result, the Au/Ag alloy NPs-Cys colorimetric assay is an uncomplicated, rapid, cost-effective, highly sensitive and selective determination method for monitoring and examining Cys.