Highly emissive cysteamine-capped gold-copper bimetallic nanoclusters (CA-AuCu NCs) with a quantum yield of 18% were synthesized via one-pot anti-galvanic reduction. The CA-AuCu NCs were characterized by HR-TEM, XPS, FTIR, MALDI-TOF mass spectrometry, DLS, and zeta potential analyses. The NCs are shown to be viable fluorescent probes for Cr(VI) ions and dopamine (DA) via quenching of the blue fluorescence, typically measured at excitation/emission wavelengths of 350/436 nm. During DA recognition, a dark brown color appears, which is distinguishable from that of Cr(VI) detection. The aggregation induced quenching due to electron transfer was demonstrated by photoluminescence, HR-TEM, FTIR, DLS, and zeta potential interrogations. In buffer of pH 7, response is linear in the 0.2 ~ 100 μM for Cr(VI) and from 0.4 ~ 250 μM for DA. The respective detection limits are 80 and 135 nM. The method was applied to the determination of both Cr(VI) and DA in (spiked) tap, lake and sea water, and in human urine samples. The low toxicity of CA-AuCu NCs was validated by the MTT assay, and their responses to Cr(VI) ions and DA was also proven by Raw 264.7 cell imaging. [Figure not available: see fulltext.].