Gold nanoclusters(AuNCs) were prepared in the presence of the nucleobases of poly-cytosine DNAs in this paper. We have demonstrated that the fluorescence of the AuNCs is quenched by thiocyanate(SCN−) through the interaction between SCN− and gold atoms. AuNCs can receive energy from nucleobases to boost their emission intensity, while in the presence of SCN−, they coordinate with Au atoms and influence the energy transfer between the nucleobases and AuNCs, leading to the fluorescence quenching. The decreased fluorescence intensity was in proportion to the concentration of SCN− in the range of 8.0×10−7–1.5×10−5 mol/L with a limit of detection of 4.2×10−7 mol/L(3σ). We further validated the practice of this probe through the detection of SCN− in natural water samples.