Two new Macroacyclic Schiff base chemosensors (L1 and L2) were synthesized by the one pot condensation reaction of 2‐[3‐(2‐formyl phenoxy)propoxy]benzaldehyde and aminophenol in a 1:2 molar ratio and were characterized by IR, NMR spectroscopy. Both Schiff bases displayed high selectivity and sensitivity towards Fe3+ over other metal ions in H2O‐DMF solution (Ag+,Cu2+, Ni2+, Zn2+, Mg+2, Mn+2, Pb+2, Co+2, Hg+2, Cr+3, Na+, Ba+2 and Cd2+) due to their structure including oxygen donor atoms. The test results showed fluorescence quenching of the fluorophores when Fe3+ was bound to the recognition units. From test results, a high selectivity for Fe3+ were discovered in this type of sensors, especially, the probe based on 2‐aminophenol exhibited more significant quenching in fluorescence intensity compared with 4‐aminophenol‐based due to its rigidity structure. In addition, the structure of ligands and their antibacterial properties was investigated.