In this paper, a microstrip sensor structure was designed and implemented to detect blood glucose levels (BGL) based on changes in resonant frequencies, achieved by creating couplings at sensitive regions. A coupled step line was used to create a sensitive region on the sensor suitable for BGL testing. Transmission matrix theory was employed for the mathematical analysis to obtain the resonant frequency. Received blood samples from 50 different patients were centrifuged, and their serums were extracted to study the performance of the microchip sensor. By placing individual serum samples (50 µl) at the sensitive region of the sensor, the scattering parameters of the sensor were measured to investigate the resonant frequency variation. In addition, the effects of basic blood parameters were analyzed based on the amount of frequency shift. High sensitivity, suitable quality (Q) factor, compact size, and acceptable reproducibility of the measured results are important features of the developed sensor.