The invention provides an electrochemical biosensor and a manufacturing method thereof. The biosensor has a front side conducting layer on the front side of a base plate and a back side conducting layer on the back side of the base plate, wherein the front side conducting layers and the back side conducting layers are different; a front side reagent block and a back side reagent block which are different are also arranged on the base plate; two cover plates respectively cover the front side reagent block and the back side reagent block; the front side reagent block and the back side reagent block comprise at least one enzyme reacting with a sample and an electron mediator respectively; the front side conducting layer is provided with three electrodes; the back side conducting layer is provided with two electrodes. According to the manufacturing method, the conducting layers are formed on the front side and the back side of the base plate through printing or magnetron sputtering; insulation layers are printed on the conducting layers; the front side and back side reagent blocks are put in the front end grooves of the insulation layer; the cover plates are attached onto the surfaces of padding layers pasted with the insulation layer and the reagent blocks. The electrochemical biosensor is used for testing two ingredients in the same sample; the blood collection of a user is reduced; the electrochemical biosensor is applicable to the detection of items of glucose, uric acid, cholesterin and the like; the electrode numbers of the front side and back side conducting layers are different, so that the recognition is easy and a test instrument can be conveniently and correctly inserted.