Green hydrogen production via water electrolysis is crucial to the strategic path toward carbon neutrality. Therefore, exploration of efficient and low-cost electrocatalysts for oxygen evolution reaction (OER) is essential due to the sluggish OER kinetics, where cobalt-iron-layered double hydroxides (CoFe-LDHs) represent a class of promising OER catalysts. Herein, a systematic study gives insights into the Co/Fe intrinsically assembled structures in CoFe-LDHs on their catalytic performances in OER, representing a new route for rational design of catalysts. Theoretical calculations suggest that the electron structure at exposed active sites can be modulated by varying the Co/Fe assembled structure and introducing oxygen vacancy. The structural characterizations of the as-synthesized CoFe-LDHs with varied Co/Fe assembled structures indicate that Co1Fe3-LDHs-Vo induces the suitable distortion in the octahedral unit structure of [CoO6] with a shorter cobalt–oxygen bonding distance and hence leads to the favorable Co active sites exposed for the formation of oxygenated intermediates. Consequently, the Co1Fe3-LDHs-Vo exhibits the unprecedented OER activity with an overpotential of 253 mV at 50 mA/cm2 and Tafel value of 26.8 mV/dec. The overall water splitting is driven by a voltage of 1.47 V at 10 mA/cm2 in 1.0 M KOH electrolyte.