This paper presents the sensitivity optimization of a microwave biosensor dedicated to the analysis of a single living biological cell from 40 MHz to 40 GHz, directly in its culture medium. To enhance the sensor sensitivity, different capacitive gap located in the center of the biosensor, below the cell position, have been evaluated with different beads sizes. The best capacitive and conductive contrasts have been reached for a gap width of 5 µm with beads exhibiting diameters of 10 and 20 µm, due to electromagnetic field penetration in the beads. Contrasts improvement of 40 and 60 % have been achieved with standard deviations in the order of only 4% and 6% for the capacitive and conductive contrasts respectively. This sensor therefore permits to measure single living biological cells directly in their culture medium with capacitive and conductive contrasts of 0.4 fF at 5 GHz and 85 µS at 40 GHz, and associated standard deviations estimated at 7% and 14% respectively.