A new type of rapid, sensitive, and selective fluorescence turn-on assay was developed for detection of histidine using a DNA-scaffolded silver nanocluster/Cu(2+) ensemble (DNA-AgNC/Cu(2+)). Cu(2+) was first bound to nucleic acids of the DNA-AgNCs forming a DNA-AgNC/Cu(2+) ensemble and then liberated into solution via the highly specific interaction between histidine and Cu(2+) in the presence of other amino acids. The fluorescence of DNA-AgNCs was greatly quenched with the addition of Cu(2+), and then the DNA-AgNC/Cu(2+) ensemble exhibited marvelous fluorescent enhancement in the presence of histidine, which showed the possibility for constructing a turn-on chemosensor of histidine. Compared to other methods, this approach promises high sensitivity, simplicity in design, convenient operation, and minimized use of organic solvents. The ultra-high selectivity demonstrated the feasibility of the assay for detecting histidine in sophisticated physical environments. The fitting range of the proposed assay is from 0 to 100 μM, with a detection limit of 1.4 μM (S/N = 3) in artificial urine samples. The protocol was evaluated by analysis of artificial urine samples with good recoveries and showed great potential for practical applications.