Oxygen vacancy is an intrinsic defect in metal oxide semiconductors and has a crucial influence on their physicochemical and electronic properties. To boost the electrochemiluminescence (ECL) efficiency of the graphite-like carbon nitride (g-C3N4), the wet-chemical-calcination method was developed to introduce an oxygen vacancy in Eu-doped g-C3N4 nanostructures for the first time. The morphology and structure characterization suggest that the Eu element was present in the matrix of the europia (Eu2O3) clusters. Because of the effect of oxygen vacancy promoting catalytic activity, the doping of Eu caused a great positive shift of onset potential and large signal amplification in cathodic ECL signals compared with pure g-C3N4. Furthermore, a novel and ultrasensitive ECL aptasensor was realized with 17β-estradiol (E2) as a prototype target by adsorption of E2 aptamer onto the Eu2O3-doped g-C3N4 (Eu2O3- g-C3N4) surface via van der Waals force. Given the specific recognition between aptamer and E2, the ECL signal decreased with the increasing concentration of E2, because the formation of E2-aptamer complex impeded the diffusion of luminophor molecules and the electrons approaching the surface of the electrode. Under the optimal cases, the as-prepared ECL aptasensor showed superior performances and also manifested outstanding selectivity toward E2. The present conceptual strategy offers a novel methodology to boost the sensitivity of the ECL sensor and promote the activity of ECL reagents.