The accumulation of pesticide residues poses a significant threat to the health of people and the surrounding ecological systems. However, traditional methods are not only costly but require expertise in analysis. An electrochemiluminescence (ECL) aptasensor was developed using chitosan and molybdenum disulfide (CTS-MoSsub.2), along with acetylene black (AB@CTS) for the rapid detection of malathion residues. Due to the weak interaction force, simple composite may lead to uneven dispersion; MoSsub.2 and AB were dissolved in CTS solution, respectively, and utilized the biocompatibility of CTS to interact with each other on the electrode. The MoSsub.2 nanosheets provided a large specific surface area, enhancing the utilization rate of catalytic materials, while AB exhibited excellent conductivity. Additionally, the dendritic polylysine (PLL) contained numerous amino groups to load abundant luminol to catalyze hydrogen peroxide (Hsub.2Osub.2) and generate reactive oxygen species (ROS). The proposed ECL aptasensor obtained a low detection limit of 2.75 × 10sup.−3 ng/mL (S/N = 3) with a good detection range from 1.0 × 10sup.−2 ng/mL to 1.0 × 10sup.3 ng/mL, demonstrating excellent specificity, repeatability, and stability. Moreover, the ECL aptasensor was successfully applied for detecting malathion pesticide residues in authentic samples with recovery rates ranging from 94.21% to 99.63% (RSD < 2.52%). This work offers valuable insights for advancing ECL sensor technology in future applications.