As a crucial indicator in food and water safety testing, the detection of Escherichia coli plays a significant role in maintaining environmental sanitation and promoting public health. Herein, based on the electrochemical activity characteristics of E. coli, we established an enhanced electrochemiluminescence aptasensor for E. coli analysis. This study presents a new method for accurate identification by utilizing a double aptamer recognition system. Specifically, a nano-cadmium sulfide (CdS) modified aptamer was used for primary labeling, while a second aptamer was immobilized on a graphene/chitosan composite electrode for re-capture. The use of two aptamers improves the accuracy of the identification process. Furthermore, the application of an electrode potential facilitates continuous electron transfer between the electrode and electrochemically active microorganisms, resulting in an enhanced electroluminescence signal in relation to the metabolic status. This strategy possesses better sensitivity, accuracy, and stability, demonstrating its potential for E. coli analysis.