Electrochemical biosensing relies on electron transport on the electrode surface. However, the limited functional area of the two-dimensional electrode prevents the qualitative breakthrough in the efficiency of electron transfer. Here, a three-dimensional electron transporter was constructed to improve the efficiency of electron transfer by using an interface-immobilized DNA hydrogel. A three-dimensional pure DNA hydrogel is constructed and used as a scaffold for electron transfer. Then, an electron mediator is embedded in the DNA hydrogel through intercalative binding, and DNAzyme with intrinsic peroxidase-like activity is introduced at the node of the hydrogel scaffold to fabricate an electrochemical biosensor. The conduction of the electron mediator in the scaffold enables the acquisition of long-distance DNAzyme catalytic signals, thereby overcoming the limitation of two-dimensional electrodes. This three-dimensional electron transporter is significant for enriching the toolbox of electrochemical biosensing and can provide potential support for the development of highly sensitive biosensors.