The invention discloses an electrochemical biosensor for detecting penicillin and a preparation method and application thereof. The electrochemical biosensor is characterized in that a working electrode is a magnetic glassy carbon electrode immobilized with MWCNTs-Fe3O4/Au-HRP-Ab. The preparation method comprises the following steps: carrying out carboxylation on a multi-walled carbon nanotube, preparing Fe3O4/Au composite nano particles, preparing a Fe3O4/Au magnetic nano particle-HRP-penicillin antibody, sequentially modifying the carbon nanotube subjected to carboxylation and Fe3O4/Au-HRP-Ab to the surface of the glassy carbon electrode of the carbon nanotube, forming the electrochemical biosensor by taking a platinum electrode as a counter electrode and taking a saturated calomel electrode as a reference electrode, immersing the electrochemical biosensor into a sample to be detected, and determining the concentration of the penicillin in the sample to be detected according to a quantitative relation between a corresponding current value and the concentration of the penicillin. The electrochemical biosensor has the advantages of high sensitivity, high selectivity, high accuracy and high detection speed.