The invention discloses a preparation method of an electrochemical sensor constructed on the basis of a nano functional material and belongs to the technical field of novel nano functional materials and biosensors. The preparation method includes: preparing a novel two-dimensional nano electrode material-the loaded bi-metal co-doped nano composite material FeCo-TiO2/MoS2, namely a two-dimensional nano composite material with an iron-cobalt co-doped titanium dioxide nano square block composited with molybdenum disulfide in situ; utilizing high biocompatibility and large specific surface area of the material to load a clenbuterol antibody; during detection, due to the fact that iron-cobalt co-doped titanium dioxide can catalyze hydrogen peroxide to generate O2 in situ to generate electrochemical signals, utilizing influence of specific quantitative combining of the antibody and antigen on electron transmission capability to correspondingly lower current strength; finally realizing construction of a biosensor adopting a mark-free electrochemical method to detect clenbuterol.