Herein, for the first time, we engineered click chemistry reaction to trigger a 3D DNA walking machine for ultrasensitive electrochemical detection of copper ion (Cu2+), which provided a convenient access to overcome the shortcomings of poor selectivity and limited amplification efficiency in traditional determination of Cu2+. Click chemistry reaction drove azido-S2 to bind with alkynyl-S1 for the formation of a walker probe on aminated magnetic polystyrene microsphere@gold nanoparticles (PSC@Au), which opened the hairpin-locked DNAzyme. In the presence of magnesium ion (Mg2+), the unlocked DNAzyme was activated to cleave the self-strand at the facing ribonucleotide site, accompanied by the release of product DNA (S3) and the walker probe. Therefore, the walker probe was able to open the adjacent hairpin-locked DNAzyme strand and then be released by DNAzyme cleavage along the PSC@Au-DNAzyme track. Eventually, the liberated single-strand S3 induced catalytic hairpin assembly (CHA) recycling, resulting in the capture of a large number of methylene blue-tagged hairpin DNA (MB-H2) on the sensor surface and significant electrochemical responses. By coupling click chemistry reaction with the dual-amplification strategy of the 3D DNA walking machine and CHA recycling, the proposed biosensor not only demonstrated high accuracy and selectivity for Cu2+ detection in real samples but also showed excellent performance for Cu2+ detection with a wide linear range of 1.0 pM to 500 nM and low detection limit of 0.33 pM. Moreover, this elaborated biosensor could be readily expanded to Mg2+ detection with a constant concentration of Cu2+, which paves a new way to apply the 3D DNA walking machine in various ion sensings.