The valorisation of glycerol using renewable electricity is recognised as an effective and attractive approach for producing high-value compounds from biomass byproducts. 2D conjugated metal–organic frameworks (2D c-MOFs) have emerged as promising electrocatalysts due to their tunable structures, high electronic conductivity, and efficient utilisation of well-defined active centres. In this study, we report the first systematic investigation of 2D c-MOFs containing Ni-X₄ (X = O or N) moieties for the glycerol oxidation reaction (GOR), examining key factors influencing both electrocatalytic activity and selectivity. In situ ¹³C electrochemical nuclear magnetic resonance and Raman spectroscopies provide insights into the GOR mechanism and confirm that Ni–O₄ sites are the primary active centres. Theoretical calculations further reveal that [Ni₃(HHTQ)₂]_n (HHTQ = 2,3,7,8,12,13-hexahydroxytricycloquinazoline) exhibits superior GOR activity due to strong adsorption of reaction intermediates and weak interlayer interactions. This work highlights the potential of 2D c-MOFs as highly efficient GOR catalysts, paving the way for the rational design of advanced electrocatalysts and contributing to the development of sustainable energy conversion and storage technologies.