We demonstrate a simple preparation protocol for the synthesis of novel adenosine monophosphate-stabilized copper nanoclusters (AMP-Cu NCs). Synthesis parameters are optimized to reach the best fluorometric signal. The prepared Cu NCs show characteristic blue emission at 430 nm, which is related to the presence of Cu13 metal cores. Three distinct lifetime components are identified and the metallic feature of the NCs is proven by X-ray photoelectron spectroscopy. The stability and the long-term storage of the AMP-Cu NCs were investigated by pH- and time-dependent measurements. Besides the synthesis and characterization, a possible liquid fluorescent assay method is also developed for the detection of the biologically important Vitamin B2 in complex real samples by with 2.01 ± 0.31 μM detection limit. For the real samples, the yeast and dietary supplements were selected, where the detected concentration of Riboflavin was in good agreement with the expected amount in these complex chemical systems.