A paper based analytical device is presented for the determination of Cr(III) and Cr(VI) using gold nanoparticles (AuNPs) modified with 2,2′-thiodiacetic acid. The modified AuNPs were characterized using UV-Vis spectrophotometry, Fourier transform infrared, dynamic light scattering, zeta potential, energy dispersive spectroscopy and transmission electron microscopy. Cr(III) ions induce the aggregation of the modified AuNPs, and the color of the nanoprobe changes from red to blue. This can be detected visually, or by colorimetry, or with a camera. No interference is observed in the presence of 19 other cations and anions. Cr(VI) (chromate) can be determined by after reduction to Cr(III) by using ascorbic acid and then quantified total Cr(III). The concentration of Cr(VI) is obtained by subtracting the concentration of Cr(III) from that of total chromium. Under optimal conditions, the ratio of the absorbances measured at 670 (blue) and 522 (red) increases linearly in the 1.0 nM to 22.1 μM chromium concentration range, with 0.66 nM (0.034 ppb) limit of detection (LOD) in solution. In case of the paper device, the linear range extends from 1.0 nM to 0.1 mM, and the LOD is 0.64 nM (0.033 ppb). The method was applied to the determination of chromium in spiked water, urine and dilutes human plasma, and results were confirmed by GF-AAS analysis. This method is highly selective, fast and portable, requires minimum volume of reagents and samples and no washing steps.