Electrochemical biosensors with ultra-high sensitivity and specificity are of great significance in the fields of environmental risk substances monitoring and biomedical detection. The key to the application of electrochemical biosensors is to construct a high biocompatibility, simple preparation process and low cost detection electrode. In this paper, three-dimensional graphene electrodes with good reproducibility were prepared by 3D printing technology, and then the morphology and oxidation groups of graphene on the surface were regulated by electrochemical oxidation. The electrochemical biosensor shows high sensitivity in the detection of environmental pollutant microcystin (MC-LR). The linear detection range is 4×10-6 μg/L to 1 μg/L, and the detection limit is 1.5×10-7 μg/L. At the same time, the electrochemical biosensor presents highly sensitivity for dopamine, heavy metal Hg2+, tetracycline and so on by changing the aptamer detection probe. This study provides a new idea for the application of electrochemical aptamer biosensors, and provides some basic data for the development of ultra-high sensitivity environmental monitoring and biomedical detection sensors.