In this work, a novel electrochemiluminescence (ECL) aptasensor was structured for the detection of four organophosphorus pesticides (OPs). Firstly, multi-walled carbon nanotubes (MWCNTs) were used to create a favorable loading interface for the fixation of tris (2, 2′-bipyridyl) ruthenium (II) (Ru (bpy)32+). At the same time, copper (core)-gold (shell) bimetallic nanoparticles (Cu@Au NPs) were synthesized in the aqueous phase for the sensor construction. Gold nanoparticles (Au NPs) could promote the electrochemiluminescence intensity of Ru (bpy)32+ with high efficiency by catalyzing the oxidation process of tri-n-propylamine (TPrA). Compared with the Au NPs, Cu@Au NPs increased the solid loading of Au NPs by virtue of the large specific surface area of copper nanoparticles (Cu NPs), which could further improve the sensitivity of aptasensor. When OPs were added, the ECL intensity was significantly reduced, and the concentration of OPs could be detected through the ECL intensity. Under the optimum conditions, the aptasensor had a wider dynamic range and ultra-low detection limit for the detection of four pesticides: profenofos, isocarbophos, phorate, and omethoate, and their detection limits were 3 × 10−4 ng/mL, 3 × 10−4 ng/mL, 3 × 10−3 ng/mL, and 3 × 10−2 ng/mL respectively (S/N = 3). The aptasensor had the merits of good stability, reproducibility, and specificity, and had a favorable recovery rate in detecting OPs residues in vegetables. This work provided an effective method for the construction of a simple, rapid, and sensitive biosensor.