Although hydrosulfide and cyanide anions play important roles in daily life that they are available in a lot of foods. However, their excess amounts contaminate water, land, and food and cause serious problems to human health. Herein, we introduce a water-soluble macrocyclic sensor based-on Calix[4]arene (MPI-Calix[4]) with dual response sites for fluorescence recognizing cyanide (CN–) and hydrogen sulfide (HS⁻) under longwave light. MPI-Calix[4] exhibits a high selectivity and sensitivity in the detection of CN– and HS⁻, where the limits of detection were as low as 0.115 and 8.12 μmol/L, respectively. The cell imaging studies shows that this probe can be easily used in the detection of CN– and HS⁻ on living cells. Full understanding of these results paved a fruitful system to improve an applicable analytical process for food safety and quality.