A new 1,3,4-oxadiazole-based fluorescence chemosensor 1, N-(2-ethoxy-2-oxoethyl)-N-(5-(2-hydroxy-3,5-di-tert-butylphenyl)-[1,3,4]oxadiazol-2-yl)glycine ethyl ester, has been designed and synthesized. Its fluorescence properties and selectivity for various metal ions were investigated in detail. A prominent fluorescence enhancement only for Zn²⁺ was found in aqueous acetonitrile solution and the response mechanism of 1 was analyzed by time-resolved fluorescence decay and DFT calculations. Furthermore, the fluorescence imaging of Zn²⁺ in living cells was successfully applied.