The limit of detection of a biological sensor is an important parameter because, when it is optimized, it allows the detection of a reduced number of biological cells and the reduction of the detection time. This parameter can be improved upon with a reduction in electrode size, but the rate of detection is similarly reduced as well. To avoid this problem, we propose a sensor matrix composed of 20 × 20 µm² coplanar square electrodes with a standard clean room manufacturing process. However, it was observed that the exposition of electrode connection tracks to the solution reduces the normalized impedance variation. In this pursuit, we propose in this paper an analysis of electrode connection tracks on the normalized impedance variation and cutoff frequencies to biological cell measurements by impedance spectroscopy. The experimental results were obtained using the E4990A Keysight impedance analyser (Keysight Technologies, Santa Rosa, CA, USA) with a frequency band ranging from 100 Hz to 12 MHz, thus allowing for good measurement accuracy. Therefore, it was found that, for the measurements between the electrodes with 9 µm of connection tracks in contact with the solution, the normalized impedance variation was from 3.7% to 4.2% for different measurements, while, for the electrodes with 40 µm of connection tracks in contact with the solution, the normalized impedance variation was from 1.8% to 2.1% for different measurements.