Microfluidic Electrochemical biosensors become more desirable since it offered an attractive replacement for the bulky and expensive analytical instruments. The design and fabrication of such sensor with high quality and optimum effective surface area is of primarily importance for more accurate result. The biosensor was designed with two different chips; the microfluidic chip which was fabricated using PDMS (Polydimethylsiloxane) and the other chip of the biosensor was fabricated using glass substrate. The microfluidic chip has the dimension of 6mm in length and 4mm width. Built-in 8 microchannels with the variation in size from 100 to 1000 µm was developed at the surface of it with inlets and outlets at the ends of each channel with the diameter of 1.5mm. An 8 electrode cells were developed at the center of each channel on a glass substrate. Each cell consisted of three electrodes, working electrode (WE), counter electrode (CE) and reference electrode (RE) with size of 100 µm, 600 µm and 600 µm respectively. The surface quality of the fabricated chips was analyzed using scan electron microscope (SEM). There wasn’t any deformation found in the wall of the channels. Moreover, the electrodes surface was of high resolution which will result in lower electrical noise during the application. A range of channels sizes was simulated using COMSOL Multiphysics simulation software. The optimal channel size was with width of 700 and depreciation on the current respond at higher sizes was observed. To this end, the fabricated sensor was desirable for the high performance of microbiological detection applications.