A new symmetrical azomethine probe, H2L, has been synthesized via a condensation reaction of N, N´-Ethylene bis (3-formyl-5-methyl salicylaldimine), with Isonicotinic hydrazide in EtOH. The anion recognition studies exhibited that H2L acts as a chemosensor for the detection of CN− and AcO− in pure DMSO and DMSO/H2O 90:10(V/V) media over other basic anions such as F−, Cl−, Br−, I−, N3 −, SO4 2−, SO3 2−, PO4 3−, H2PO4 −, HSO4 −, NO2 −, and NO3 −. The ability of the H2L for rapid colorimetric sensing of CN− and AcOˉ was studied using UV–Vis, and 1H NMR titration methods. The detection limits of H2L toward CN− and AcO− in the semi-aqueous medium were found to be 2.12 and 2.63 μM, respectively, which are lower than the LOD of some existing azomethine probes. Effectively, H2L was used to qualitatively and quantitatively detect cyanide ions in the human-poisoned blood and some of the agro-products containing cyanogenic samples without relying on expensive instruments.