Fluorescent zinc complexes have recently attracted a lot of interest owing to their vast applications in cellular imaging. We report the synthesis as well as physical, chemical and biological studies of a novel zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone), [Zn (GTSC)](3), complex. As compared with the well-studied zinc biacetylbis(4-methyl-3-thiosemicarbazone), Zn(ATSM), complex, which was used as a reference, [Zn(GTSC)](3) had 2.5-fold higher fluorescence. When cellular fluorescence was measured using flow cytometry, we observed that [Zn(GTSC)](3) had 3.4-fold to 12-fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy results showed that [Zn(GTSC)](3) appeared to have a nuclear localization within 30 mm of addition to MCF7 cells. Moreover, [Zn(GTSC)](3) showed minimal cytotoxicity compared with Zn(ATSM), suggesting that [Zn(GTSC)](3) may be less deleterious to cells when used as an imaging agent. Our data suggest that the novel [Zn(GTSC)](3) complex can potentially serve as a biocompatible fluorescent imaging agent for live cells.