The invention belongs to the technical fields of novel nanomaterials, immunoassay and biosensing detection and provides a preparation method and application of an mHPBs-Hemin/rGO marked electrochemical immunosensor. According to the preparation method, a mesoporous hollow Prussian-blue nanocube spaced hemin functional graphene porous composite is used as a current signal amplifying mechanism and introduced into construction of the electrochemical immunosensor for the first time, a current response signal is amplified efficiently, and quantitative and sensitive detection of aflatoxin is realized. The electrochemical immunosensor is easy and convenient to operate, low in cost, high in sensitivity and wide in detection range and has important scientific significance and application value forquantitative and sensitive detection of aflatoxin.