The CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) with strong exciton confinement have manifested themselves as competitive light-emitting materials in electrochemiluminescence (ECL). However, cathodic ECL generation by these QDs requires the injection of electron and hole from solid electrode and electrogenerated radicals (for example SO₄^•−), which is inevitably influenced by not only the inorganic structure of QDs but also the organic ligands on the surface. In this work we aimed at studying the impact of surface organic ligands on ECL performance of CdSe/CdS/ZnS QDs. When changing the surface ligand from oleate to acetate, we phenomenologically observed the positive shift of ECL onset potential by ca. 200 mV and the increase of ECL intensity by ~ 100 times, suggesting that a short ligand is more favorable for ECL generation. To further comprehend the ligand effect, we measured the charge injection kinetics using potential-modulated, time-resolved photoluminescence, and thin-layer spectroelectrochemistry techniques. The electron and hole injection into QDs were found to be accelerated by 2–20 times if shortening the ligand from oleate to acetate, confirming the significant impact of surface ligands on ECL performance of QDs. The study is expected to provide guidance on how to design surface functionalized QDs for specific applications such as ECL immunodiagnosis, photocatalysis, and photovoltaics.