By dissolution of Au NPs, new polyacetylene-functionalized Au NPs were successfully developed as “turn-on” fluorescent probes for the detection of cyanide sensitively and selectively. The lowest concentration to quantify cyanide ions could be down to 3.0 × 10−6 M, and other common anions including AcO−, Br−, C2O42−, Cl−, CO32−, F−, HCO3−, NO2−, NO3−, PO43−, S2O82−, SCN−, SO32−, SO42−, HPO42−, and P2O74− had almost no influence. Furthermore, several real water samples spiked with cyanide, including local groundwater, tap water, boiled water, and lake water, were analyzed with RSD values less than 4%, and the experimental results demonstrated that our developed sensing system worked well in the above water samples.