Electrochemical measurements were conducted using a custom-made chip holder. This holder contained a sample well sealed to the chip with a fluoroelastomers O-Ring and a lid fastened with screws for secure attachment to the chip. In this work, the detection of glucose was based on measuring the H2O2 by-product produced enzymatically. Thus, the modified sensor electrode surface (Pt-B/o-PD/β-CD) was analyzed in different concentrations of H2O2 in the absence of GOx, to ensure the possible detection of H2O2 at the microband electrode arrays. CV and CA electrochemical techniques were carried out to measure the pH level ranging from pH 5 to 8 in bare Au electrode and known concertation of glucose within physiologically relevant range in 10 mM PB (pH 7.4 & pH 5.5) at modified electrode surface of Pt-B/o-PD/β-CD/GOx, respectively. Furthermore, electrochemical pH control was undertaken sequentially by CV and CA scans for both the pH and glucose biosensor. The limits of detection (LODs) were determined by calculating three times the standard deviation for the blank solution (PB buffer solution) and dividing it by the slope of the corresponding calibration curve. The biosensor's selectivity was evaluated against a mixture of interfering species commonly found in saliva. The interferents included 0.5 mM of each AA, UA, D-Fruc, and Ace, all assessed in the presence of a 2 mM and 5 mM glucose concentration in 10 mM PB (pH 7.4) which were measured using CA method. Stability of the biosensor was examined by measuring a 4 mM glucose concentration for 85 days. In addition, both pH level and spiked glucose concentrations were measured using artificial saliva along with pH control by using CVs and CAs, respectively. Finally, the biosensor was tested in eight different calf saliva samples of known disease status to confirm the approach for disease detection. The pH was controlled and optimized to pH 5.5 for the measurements of the calves saliva to find out the actual level of the glucose concentrations in saliva. Additionally, a commercial glucometer was used to check the glucose level in real calf saliva samples. The OriginPro 2021 software was utilized to compute and analyzed the equation for the linearity range and associated slopes.