A rhodanine-based fluorescence chemosensor HTO, 3-((2-hydroxybenzylidene)amino)-2-thioxothiazolidin-4-one, was prepared by the reaction of a 3-aminorhodanine and a salicylaldehyde for detecting In3+. HTO could detect In3+ with turn-on fluorescence in a near-perfect aqueous media. Detection limit was calculated to be 1.69 μM and the binding feature of HTO with In3+ was determined to be 1:1 by Job plot and ESI-mass. The sensing behavior of HTO toward In3+ was proposed by using photophysical experiments, ESI-mass and theoretical calculations. In a very rare case, HTO was successfully applied to detect In3+ in live organisms (Hela cells and zebrafish).