A new method for the determination of traces of mercury in environmental and biological samples is described. The present methodology combines determined using a hydride generation-atomic absorption spectrometry (HG-AAS) with pre-concentration/separation of the analyte on dithizone-anchored organobentonite (D-O-bentonite). Optimal experimental conditions for the adsorption of the Hg, including pH, contact time, eluent concentration and volume and co-existing ions have been studied. The result showed that the mercury could be adsorbed on the D-O-bentonite. The adsorbed quantitively was affected by the pH value of medium and contact time. In the medium of pH 5.0, the adsorption time was 15 min, and capacity of adsorption was 23.2 mg·g-1. The mercury adsorbed on the sorbent could be completely eluated by using 3 mol·L-1 HCl. The adsorption agent has been applied to the pre-concentration/separation of mercury in surface water and human hair samples with satisfied results.