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Developing low-cost and multiplexing electrochemical (EC) devices for bioassay is imperative. Herein, a polymer-based EC device, named EC 6-well plate, was proposed and fabricated using a non-photolithography method. Polyethylene terephthalate glycol (PETG) was used as a substrate and laser-cut polyester (PET) film was used as a mask for patterning the electrodes. The diameter of the working electrode (WE) was 900 μ m, and each WE-modifying step only requires 1 μ l of reagent. Acrylic mold with wells (60 μ l) was bonded to the PETG substrate. Miniaturization of reference electrodes (RE) was discussed. The solid-state Ag/AgCl RE-based three-electrode system, the Au three-electrode system (3E), and Au two-electrode system (2E) were prepared and employed to develop an immunosensor for toxin B detection. Differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were applied to test the stability of the EC immunosensor. The solid-state Ag/AgCl RE-based system showed a standard deviation of open circuit potential (OCP) of 4.6 mV. The 3E system and 2E system showed the standard deviations of OCP of 0.0026 mV and 0.32 mV, respectively. It revealed that the EC 6-well plate with the 3E system is excellent for developing an EC immunosensor.