Copper ion (Cu(2+)) plays an important role in many biological reactions, and a suitable level of Cu(2+) is necessary for the regular metabolism of life. Thus developing a sensitive and simple method for determination of Cu(2+) is essential. Here, a novel and sensitive Cu(2+) sensor was developed based on detecting the average hydrodynamic diameter of AuNPs by using dynamic light scattering (DLS). Cu(2+)-specific DNAzyme was double-strand and could not adsorb on the surface of AuNPs, accordingly AuNPs aggregation would occur with the addition of NaCl. However, Cu(2+) could cleave DNAzyme and release single-stranded DNA (ssDNA) fragments, which could adsorb on the surface of AuNPs and prevent them from aggregation. Such differences in DNA adsorption ability on AuNPs before and after the addition of Cu(2+) affected the disperse state of AuNPs directly, and then affected their average hydrodynamic diameter, which could be detected with the DLS technique. Based upon the above mentioned principle, detection of Cu(2+) could be realized over the range from 100 pM to 2.0 nM, with a linear regression equation of D = 306.73 - 89.66C (C: nM, R = 0.9953) and a detection limit of 60 pM (3δ/slope). Moreover, satisfactory results were obtained when the assay was applied in the detection of Cu(2+) in water samples.