A sensitive, stable and stereoselective electrochemiluminescence (ECL) sensor has been designed to enantioselectively discriminate proline enantiomers by immobilizing Ru(bpy)₃²⁺–gold nanoparticles (Ru–AuNPs) and β-cyclodextrin–reduced graphene oxide (β-CD–rGO) on glassy carbon electrode. More Ru(bpy)₃²⁺ could be immobilized on the surface of electrode stably via preparing Ru–AuNPs and better stereoselectivity could be introduced to the sensor via the synthesis of β-CD–rGO. When the developed sensor interacted with proline enantiomers, obvious difference of ECL intensities towards L- and D-proline was observed, and a larger intensity was obtained from D-proline. As a result, ECL technique might act as a promising method to chiral recognition of amino acids enantiomers or chiral drugs.