Due to the fascinating properties such as high porosity, large surface areas, and tunable chemical components, metal-organic frameworks (MOFs) have emerged in many fields including catalysis, energy storage, and gas separation. However, the intrinsic electrical insulation of MOFs severely restricts their application in electrochemistry. Here, we synthesize a series of 2D conductive MOFs (cMOFs) through tuning the structure with atomic precision using simple hydrothermal methods. Various electroactive probes are used to reveal the structure-property relationships in 2D cMOFs. Then, we demonstrate the first exploration and implementation of 2D cMOFs toward the construction of electrochemical biosensors. In particular, the biosensor based on Cu3(tetrahydroxy-1,4-quinone)2 [Cu3(THQ)2] displays a remarkably improved electrocatalytic performance at a much lower potential. The mechanism study reveals the essential role of charge-transfer interactions between the dense catalytic sites of Cu3(THQ)2 and analytes. Furthermore, the Cu3(THQ)2-based biosensor demonstrates robust anti-interference capability, good stability, fast response speed, and an ultralow detection limit for paraoxon. These promising results indicate the great potential of cMOFs in biomedical, food safety, and environmental sensing applications.