This study presents a convenient and homogeneous method for the selective detection of hydrogen cyanide in cyanogenic glycoside-containing plants using polysorbate 40-stabilized gold nanoparticles (PS 40-AuNPs). Neutral PS 40 molecules stabilized citrate-capped AuNPs in a high-salinity solution. When the plant tissue is damaged, β-glucosidase rapidly hydrolyzes cyanogenic glycosides to produce hydrogen cyanide. The addition of the released hydrogen cyanide to a solution of PS 40-AuNPs resulted in the formation of AuCN(s) on the NP surface and Au(CN)2− in an aqueous solution. The removal of PS 40 molecules from the NP surface rendered the AuNPs unstable in a high-salinity solution, leading to NP aggregation. We used surface-assisted laser desorption/ionization time-of-flight mass spectrometry to detect the formation of AuCN(s). This probe was also successfully used to determine the total cyanide in cassava roots, peach kernels, and loquat kernels. When the three cyanogenic glycoside-containing plants were separately heated, this probe was capable of monitoring the removal of hydrogen cyanide during heating. In addition, the use of thiosulfate to detoxify the three cyanogenic glycoside-containing plants was evaluated using PS 40-AuNPs.