In this paper, we have developed a label-free fluorescence method for sensitive detection of mercury ion in homogeneous solutions. The assay exploits the fluorometric signal change of hoechst33258 when DNA probe conformational transformed from a random coil to a hairpin structure upon binding to Hg2+. Under the optimum conditions, the system exhibits a dynamic response for Hg2+ from 10 nM to 2 μM with a detection limit of 1nM. It is a simple, selective, and sensitive method for Hg2+ detection.