Trace Zn(2+) and Cu(2+) in living cells play important roles in the regulation of biological function. It is significant to simultaneously detect the cellular Zn(2+) and Cu(2+). Here, we present a novel two-color fluorescence nanoprobe based on the DNAzymes for simultaneous imaging of Zn(2+) and Cu(2+) in living cells. The probe consists of a 13 nm gold nanoparticle, DNAzymes that are specific for Zn(2+) and Cu(2+), and the substrate strands labeled with fluorophores at the 5' end and quenchers at the 3' end. The fluorescence of the fluoreophores is quenched both by the gold nanoparticle and the quencher. After the nanoprobes are transferred into the cells, the substrate strands would be cleaved in the presence of the Zn(2+) and Cu(2+) target, resulting in disassociation of the shorter DNA fragments containing fluorophores, which produce fluorescence signals correlated with the location and concentration of the Zn(2+) and Cu(2+). The nanoprobe exhibits high specificity, nuclease stability, and good biocompatibility. Moreover, the nanoprobe can simultaneously monitor the cellular Zn(2+) and Cu(2+) with an on-site manner, providing the information on localization and concentration of targets, which is significant to further research the Zn(2+)- and Cu(2+)-relative cellular events and biological process. The proposed method has shown great potential in the detection of multiple metal ions in living cells, which may help us to better understand the function of metal ions in the fields of biochemistry, molecular biology, and cellular toxicology.