We have described a simple and reliable colorimetric method for the sensing of biothiols such as cysteine, homocysteine and glutathione in biological samples. The selective binding of chitosan capped silver nanoparticles to biothiols induced aggregation of the chitosan–Ag NPs. But the otheramino acids that do not have thiol group cannot aggregate the chitosan–Ag NPs. Aggregation of chitosan–Ag NPs has been confirmed with UV–vis absorption spectra, zeta potential and transmission electron microscopy images. Under optimum conditions, good linear relationships existed between the absorption ratios (at A500/A415) and the concentrations of cysteine, homocysteine, and glutathione in the range of 0.1–10.0 μM with detection limits of 15.0, 84.6 and 40.0 nM, respectively. This probe was successfully applied to detect these biothiols in biological samples (urine and plasma).