The classical electrochemiluminescence (ECL) reagent Ru(bpy)32+ was first doped into CdSe QDs to prepare novel dual-potential color-resolved luminophore Ru-CdSe QDs. Ru-CdSe QDs emitted a strong red ECL signal at a positive potential with coreactant TPrA and a strong green ECL signal at a negative potential with coreactant K2S2O8. As a proof-of-concept application, this work introduced Ru-CdSe QDs into a dual-channel closed bipolar electrode (CBPE) system to construct an ECL biosensor for simultaneous detection of chloramphenicol (CAP) and kanamycin (KAN). Ru-CdSe QDs were dropped on BPE holes A and B for ECL emission. Cobalt single-atom catalysts (Co-N-C SACs) had superior electric double layer (EDL) performance and conductivity. It could greatly promote the electron transfer of the CBPE system and realize ECL signal amplification. Based on this characteristic, Co-N-C SACs were introduced into BPE hole C and driving electrode hole D, respectively, using the CAP and KAN split dual-aptamer sandwich strategy. During positive potential scanning, the polarity of BPE hole A was anode, and Ru-CdSe QDs emitted a red ECL signal. With the increase of CAP concentration, abundant Co-N-C SACs were introduced to the electrode surface. The positive potential ECL signal was increased for CAP detection. During negative potential scanning, the polarity of the BPE hole B was cathode, and Ru-CdSe QDs emitted a green ECL signal for KAN detection. Finally, a zero-background spatial-potential color-resolved CBPE-ECL biosensor was developed for dual-mode detection of CAP and KAN. This work explored a novel ECL luminophore to construct a CBPE-ECL sensor, which greatly facilitated the development of the ECL assay.