The invention discloses a preparation method and application of an electrochemical malathion biosensor based on a bi-metal in-situ composite two-dimensional nanomaterial and belongs to the technical field of novel nano functional materials and biosensors. A novel flaky magnetic nano light-sensitive material FeMn-N@TiO2 is prepared for the first time; through good bio-compatibility, large specific area and ferromagnetism of the material, a malathion antibody is loaded, then horse radish peroxidase is fixed through the interlinking effect of glutaraldehyde, in the detection process, due to the fact that horse radish peroxidase can catalyze hydrogen peroxide, electrochemical signals are generated, through the influences of specific quantitative combination of the antibody and an antigen on electron transmission capacity, the current intensity is correspondingly reduced, and finally the electrochemical biosensor which is low in cost, high in sensitivity, good in specificity, rapid in detection and easy to prepare and detects malathion is prepared.