The invention relates to an electrochemical biosensor for organic phosphorus pesticide detection and a preparation method of the electrochemical biosensor. The preparation method comprises the following steps: polymerizing thionine to the surface of a silk-screen printing carbon electrode so as to modify polythionine on the silk-screen printing carbon electrode, fixing acetylcholin esterase and a carboxylation multi-wall carbon nanotube on the surface of the polythionine modified silk-screen printing carbon electrode through chitosan gel, and drying, thereby manufacturing the silk-screen printing carbon electrode, that is, the working electrode of the electrochemical biosensor. The invention provides the electrochemical biosensor which is applied to organic phosphorus pesticide detection and is relatively good in stability, relatively high in sensitivity and relatively low in cost; the invention further provides the preparation method of the electrochemical biosensor for organic phosphorus pesticide detection, the cost of the preparation method is relatively low, and an electrochemical biological bed manufactured by using the electrochemical biosensor is relatively good in stability and relatively high in sensitivity.