This article presents a high-sensitivity, quantified, linear, and mediator-free resonator-based microwave biosensor for glucose sensing application. The proposed biosensor comprises an air-bridge-type asymmetrical differential inductor (L) and a center-loaded circular finger-based inter-digital capacitor (C) fabricated on Gallium Arsenide (GaAs) substrate using advanced micro-fabrication technology. The intertwined asymmetrical differential inductor is used to achieve a high inductance value with a suitable Q-factor, and the centralized inter-digital capacitor is introduced to generate an intensified electric field. The designed microwave sensor is optimized to operate at a low resonating frequency that increases the electric field penetration depth and interaction area in the glucose sample. The microwave biosensor is tested with different glucose concentrations (0.3–5 mg/ml), under different ambient temperatures (10–50 °C). The involvement of advanced micro-fabrication technology effectively miniaturized the microwave biosensor (0.006λ0 × 0.005λ0) and enhanced its filling factor. The proposed microwave biosensor demonstrates a high sensitivity of 117.5 MHz/mgmL-1 with a linear response (r2 = 0.9987), good amplitude variation of 0.49 dB/mgmL-1 with a linear response (r2 = 0.9954), and maximum reproducibility of 0.78% at 2 mg/mL. Additionally, mathematical modelling was performed to estimate the dielectric value of the frequency-dependent glucose sample. The measured and analyzed results indicate that the proposed biosensor is suitable for real-time blood glucose detection measurements.