A short ssDNA (Apt-21) rationally truncated from the parent 100 nt As(III) aptamer was used for colorimetric determination of As(III). Apt-21 serves dual functions, i.e., recognition of trace As(III) and regulation of AuNPs dispersion by surface attachment, while gold nanoparticles (AuNPs) functioned as colorimetric signal reporters. Under the optimal conditions, the ratio of the absorbance at 650 nm to 520 nm (A650/A520) of AuNPs changed proportionally with increasing concentration of As(III), which showed a linear relationship within the concentration ranges 1-30 ppb and 30-100 ppb with a detection limit of 0.18 ppb. The feasibility of this assay was demonstrated by determining As(III) in spiked water samples with mean recoveries ranging from 96.5-107.1%. Schematic representation of colorimetric detection of As(III) based on the short ssDNA (Apt-21) and gold nanoparticles (AuNPs).