A novel fluorescent detection strategy for simultaneous detection of Cu2+, Pb2+ and Mg2+ based on DNAzyme branched junction structure with three kinds of DNAzymes and graphene oxide (GO) was presented. Three fluorophores labeled DNA sequences consisted with enzyme-strand (E-DNA) and substrate strand (S-DNA) were annealed to form DNAzyme branched junction structure. In the presence of target metal ion, the DNAzyme was activated to cleave the fluorophore labeled S-DNA. The S-DNA fragments were released and adsorbed onto GO surface to quench the fluorescent signal. The detection limit was calculated to be 1 nM for Cu2+, 200 nM for Mg2+, and 0.3 nM for Pb2+, respectively. This strategy was successfully used for simultaneous detection of Cu2+, Mg (2+) and Pb2+ in human serum. Moreover, it had potential application for simultaneous detection of multiple metal ions in environmental and biological samples. (C) 2017 Elsevier B.V. All rights reserved.