Well-dispersed Ag 2S nanocrystals with size of about 20~30 nm were prepared in distilled water at room temperature with the assistance of imidazoline surfactant quaternary ammonium salt of 2-undecyl-1-dithioureido-ethyl-imidazoline (SUDEI) prepared in-house. The products were characterized by TEM, XRD, and FT-IR, respectively. The influence of SUDEI concentration on the dispersion of Ag 2S products was briefly discussed. Furthermore, the obtained Ag 2S nanocrystals product was applied into DNA hybridization analysis, and the results indicated that the detection limit of target ssDNA was up to pmol/L, showing that the DNA probe labeled with Ag 2S nanoparticles is of promising application value in electrochemical DNA detection analysis and biosensors.