A novel anthracene based fluorescent probe containing benzothiazole group (BFA) was designed and synthesized. The cation binding properties of BFA were studied in the presence of various cations. Both UV–vis and fluorescence spectroscopic studies indicated that BFA was highly sensitive and selective toward trivalent cations (Cr3+ and Fe3+), while there was no response to monovalent, divalent cations and also Al3+ ion. The interaction of BFA with Cr3+ and Fe3+ caused a significant enhancement in emission intensity due to the combination of a unique anthracenyl static excimer formation, the restricted C=N isomerization and the suppression of highly efficient photoinduced electron transfer (PET) process. The detection limits of BFA for Cr3+ and Fe3+ were 0.46 and 0.45 µM, which presented a pronounced sensitivity toward Cr3+ and Fe3+, respectively. Also, possible utilization of BFA as bio-imaging fluorescent probe to detect Cr3+ and Fe3+ in human prostate cancer cell lines was observed by confocal fluorescence microscopy.