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A new "turn-on" fluorescent probe Py based on rhodamine and piperonaldehyde was designed and synthesized for detecting Fe3+ in cells. The free probe Py was non-fluorescent. While only upon addition of Fe3+, the significant increase of the fluorescence and color were observed which could be visible directly by "naked-eye". The probe Py shows high selectivity and sensitivity for Fe3+ over other common metal ions in EtOH-H2O (3/2, v/v) mixed solution. The association constant and the detection limit were calculated to be 4.81 × 104 M−1 and 1.18 × 10−8 mol/L respectively. The introduction of piperonaldehyde unit could increase probe rigidity which could enhance its optical properties. Meanwhile, the binding mode between Py and Fe3+ was found to be a 1:1 complex formation. The density functional theory (DFT) calculations were performed which would further confirm the recognition mechanism between probe Py and Fe3+. In addition, the probe has been proved to be reversible for detecting Fe3+. Moreover, the probe Py was used to detect Fe3+ in cells successfully. [ABSTRACT FROM AUTHOR]