The invention discloses a preparing method of an electrochemiluminescence Clenbuterol sensor based on a magnetic two-dimensional nanocomposite, and belongs to the technical field of novel nanometer functional materials and biosensors. The preparing method comprises the steps that firstly, a novel bi-metal co-doped two-dimensional manometer material FeMn-TiO2/g-C3N4 is prepared, and a Clenbuterol antibody is loaded by using the good biocompatibility and the large specific area of the material; secondly, horse radish peroxidase is fixed through the cross-linking action of glutaraldehyde, in the detection process, due to the fact that horse radish peroxidase can catalyze hydrogen peroxide to generate oxygen in situ and is subjected to an electrochemical reaction with a base solution to generate high and stable electrochemiluminescence signals, then the influence on the electron transmittability by the specific and quantitative combination of the antibody and an antigen is used, the electrochemiluminescence signals are reduced correspondingly, and finally the Clenbuterol detecting electrochemiluminescence biosensor low in cost, high in sensitivity and specificity, fast in detection and easy to prepare is obtained.