Vesicles as a typical interface-rich microenvironment can promote the reaction rate and the intermediate stability, which are promising for introduction in electrochemiluminescence (ECL) signal amplification. In this work, a kind of multilamellar vesicle obtained from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) was used to modify the electrode surface. The AOT vesicle-modified microenvironment could significantly enhance the ECL performances for the luminol/O 2 system in a neutral medium. The mechanism study demonstrated that the nanoscale multilamellar vesicles could maintain the vesicle structure on the electrode surface, which substantially improved the electron transfer and reaction rate, luminescence efficiency of the excited-state 3aminophthalate anion, and stability of the superoxide anion radical. Alternatively, such a multifunctional microenvironment was also able to enhance the ECL signals from the tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy) 3 2+ )/tripropylamine (TPrA) system. Moreover, another dodecyl dimethyl(3-sulfopropyl) ammonium hydroxide inner salt (DSB)-based vesicle was constructed to further verify the versatility of the vesicle-modified microenvironment for ECL signal amplification. Our work not only provides a versatile microenvironment for improving the efficiency of various ECL systems but also offers new insights for the microenvironment construction using the ordered assemblies in ECL fields.