The invention provides an electrochemical nanometer immunosensor and a preparation method and application thereof. According to the immunosensor, nanogold and horse radish peroxidase are fixed to the surface of an electrode through sodium alga acid used as a bridging agent. An anti-glutathione sulfurtransferase antibody is adsorbed to the nanogold. The preparation method comprises the steps that 1, the electrode is pretreated, and coated with a mixed solution composed of a sodium alga acid solution, nanogold sol and a horse radish peroxidase solution; 2, the electrode continues to be coated with the anti-glutathione sulfurtransferase antibody; 3, confining liquid is used for confining, and therefore the electrochemical nanometer immunosensor is obtained. The electrochemical nanometer immunosensor is not affected by the turbidity and color of sample, does not need to pretreat the sample through purification, enrichment and the like, is easy to operate, high in detection sensitivity, strong in specificity and capable of being used for rapidly and quantitatively detecting the horse radish peroxidase, and has the good application prospect.