2D metal-organic frameworks (MOFs) are considered as promising electrochemical sensing materials and have attracted a lot of attention in recent years. Compared with bulk MOFs, the construction of 2D MOFs can increase the exposure of active sites by obtaining a larger surface area ratio. Herein, a facile one-pot hydrothermal synthesis of pyridine-regulated lamellar Ni-MOFs with ultrathin and well-defined 2D morphology is described. Compared with the bulk structure, the 2D lamellar Ni-MOF has higher surface area and active site density, showing better electrochemical glucose sensing performance. The 2D lamellar Ni-MOF exhibits a fast amperometric response of less than 3 s and a high sensitivity of 907.54 µA mm -1 cm -2 toward glucose with a wide linear range of 0.5-2665.5 µm. Furthermore, the 2D lamellar Ni-MOF also possesses excellent stability and reproducibility, and can be used to detect glucose with high accuracy and reliability in different environments.