The invention belongs to the technical field of immunoassay and biosensation and provides a preparation method of an immunosensor based on sea urchin shaped core-shell type gold @ palladium nanospheres. Aminated graphene carried gold nanorods are adopted as a substrate, and a sea urchin shaped core-shell type gold @ palladium nanosphere marked human epididymal protein 4 is adopted as a detection antibody solution to construct an electrochemical immunosensor, quantitative detection on an ovarian cancer marker is achieved, and the immunosensor has the advantages of being good in specificity, high in sensitivity, low in detection limit, and the like, and has great scientific significances and application values for detection on ovary diseases.