A new bis(thienyl)imidazole-based fluorescent organic molecular sensor (TIBIT) was prepared and fully characterized by NMR spectroscopy, mass spectrometry and single crystal X-ray diffraction. The sensing application of TIBIT towards metal ions in solution was investigated using UV–vis, fluorescence, and surface-enhanced Raman spectroscopy (SERS). In the organic solvent THF, TIBIT showed excellent sensitivity towards Cu(II) with a detection limit of 10 pM, whereas in aqueous media, aggregation-caused quenching (ACQ) was observed. Using a SERS substrate consisting of Au-deposited long-range ordered crystals (Au-LROCs) of polystyrene colloids and a uniform deposition of a TIBIT monolayer via bonding through the sulfur atoms of the thiophene rings, ACQ was overcome enabling the detection of trace amounts of Cu(II) in aqueous media. Using this novel approach, a detection limit of 13 nM was found, considerably lower than the recommended safe level of 20 µM regulated by the United States Environmental Protection Agency for Cu2+ in drinking water, thus demonstrating the potential of TIBIT for monitoring Cu(II) levels in water samples.