A π-conjugated fluorescent probe, thiophene-2carboxylic acid{5-[(thiophene-2-carbonyl)-hydrazonomethyl]thiophen-2-ylmethylene}hydrazide (1), comprising a thiophene− thiophene conjugate, has been designed, synthesized, and characterized by elemental analysis, FT-IR, 1 H and 13 C NMR, and ESI-MS techniques. The presence of N-, O-, and S-atoms enables 1 to be a good cation-chelating agent. Probe 1 is highly selective and ultrasensitive toward detection of biologically crucial heavy transition-metal ions Zn 2+ , Cd 2+ , Cu 2+ , and Hg 2+ ions, and found best for Zn 2+ in aqueous-ethanol media. Probe 1 gives a "turn-on" fluorescence response toward Zn 2+ and Cd 2+ and a "turnoff" response in the presence of Cu 2+ and Hg 2+ . The association constants and detection limits determined for Zn 2+ and Cd 2+ are higher (10 8 −10 7 M −1 , 0.80−10 ppb) relative to those of Cu 2+ and Hg 2+ (10 5 M −1 , >100 ppb). Job's plot, ESI-mass, and DFT studies suggested a 2:3 binding stoichiometry between 1 and Zn 2+ /Cd 2+ whereas a 1:1 binding stoichiometry between 1 and Cu 2+ /Hg 2+ . The present report deals with the development of an N,O-chelating probe that recognizes four competing cations following the selectivity order Zn 2+ > Hg 2+ > Cu 2+ > Cd 2+ and the sensitivity order Zn 2+ > Cd 2+ > Cu 2+ > Hg 2+ . To have mechanistic insights, the reactions of 1 with representative metal ions Zn 2+ and Hg 2+ led to the formation of complexes [Zn 3 (1) 2 ] and [Hg 2 (1) 2 ] that were characterized and studied using ESI-MS and DFT calculations. In order to explore the possibility of practical applications of 1, the detection of Zn 2+ and Hg 2+ has been successfully accomplished in real water samples.