Cysteine is one of the important amino acids that is involved in various physiological processes, food industries, pharmaceuticals, and personal care. It also serves as a biomarker for some diseases. The large use of cysteine necessitates rapid, cheap, and accurate determination of cysteine in a range of samples. Although many techniques have been employed for the detection of cysteine, they suffer from limitations that make them unsuitable for routine analysis. Here we report on a cheap colorimetric method using biosynthesized silver nanoparticles (AgNPs) as nanozymes. The AgNPs were characterized by UV/visible spectrophotometry, scanning electron microscopy (SEM), and surface-enhanced Raman spectroscopy (SERS). The AgNPs exhibit peroxidase-like activity usingo-phenylenediamine (OPD) as a chromogenic reagent. The lowKmvalues observed for OPD and H2O2(0.9133 and 61.56 mM respectively) show strong affinity of the substrates to AgNPs. The peroxidase-like activity of AgNPs, however, was inhibited on the addition of cysteine. The results show that the absorption intensity of the oxidized OPD decreased linearly with the concentration of cysteine in the range of 0.5–20 μM. The limit of detection (LOD) in this linear range was found to be as low as 90.4 nM. The recovery from urine sample (spiked with cysteine) analyses demonstrated the feasibility of the method in real sample application. From our findings, we anticipate that our method can be applied for the analysis of cysteine in various samples.