Constructing high-performance glucose sensors is of great significance for the prevention and diagnosis of diabetes, and the key is to develop new sensitive materials. In this paper, a series of Ni2Co1-L MOFs (L = H2BPDC: 4,4 & PRIME;-biphenyldicarboxylic acid; H2NDC: 2,6-naphthalenedicarboxylic acid; H2BDC: 1,4-benzenedicarboxylic acid) were synthesized by a room temperature stirring method. The effects of metal centers and ligands on the structure, compositions, electrochemical properties of the obtained Ni2Co1-L MOFs were characterized, indicating the successful preparation of layered MOFs with different sizes, stacking degrees, electrochemical active areas, numbers of exposed active sites, and glucose catalytic activity. Among them, Ni2Co1-BDC exhibits a relatively thin and homogeneous plate-like morphology, and the Ni2Co1-BDC modified glassy carbon electrode (Ni2Co1-BDC/GCE) has the highest electrochemical performance. Furthermore, the mechanism of the enhanced glucose oxidation signal was investigated. It was shown that glucose has a higher electron transfer capacity and a larger apparent catalytic rate constant on the Ni2Co1-BDC/GCE surface. Therefore, tunable non-enzymatic glucose electrochemical sensing was carried out by regulating the metal centers and ligands. As a result, a high-sensitivity enzyme-free glucose sensing platform was successfully constructed based on the Ni2Co1-BDC/GCE, which has a wide linear range of 0.5-2899.5 & mu;M, a low detection limit of 0.29 & mu;M (S/N = 3), and a high sensitivity of 3925.3 & mu;A mM(-1) cm(-2). Much more importantly, it was also successfully applied to the determination of glucose in human serum with satisfactory results, demonstrating its potential for glucose detection in real samples.