We have developed a new dual-tip glucose sensing scanning electrochemical microcopy (SECM) probe by covalently immobilizing the glucose oxidase (GOD) enzyme onto an ultramicro electrode (UME) to measure the local glucose consumption of Streptococcus mutans (S. mutans) biofilms. GOD was immobilized on a novel enzyme immobilization matrix of functionalized multiwalled carbon nanotubes (f-MWCNTs) and 1-butyl-4-methylpyridinium hexafluorophosphate (ionic liquid/IL) packed into the etched Pt UME. The highly selective GOD-based SECM tip showed a high current density of 94.44 (±18.55) μA·mM-1·cm-2 from 0.10 to 1.0 mM at 37 °C as a result of the synergetic effects of f-MWCNTs and ionic liquid. The detection limit of the new 25 μm diameter glucose sensor is 10.0 μM with a linear range up to 4.0 mM. The sensor was successfully used to quantify the rate of glucose consumption of S. mutans biofilms in the presence of sucrose. S. mutans catabolizes both glucose and sucrose, producing lactic acid, reducing the local pH, and causing dental caries. With sucrose, S. mutans produces exopolysaccharides to enhance bacterial adhesion on the tooth surface; subsequent lactic acid production reduces the local pH, resulting in dental caries. Because of the high selectivity of the sensor, we were able to quantify glucose consumption in the presence of sucrose. S. mutans preferentially consumed sucrose in a mixed diet of both sucrose and glucose. Furthermore, using this unique fast-response (∼2 s) glucose sensor, we were for the first time able to map the distribution of the glucose consumption profile in the local environment of S. mutans biofilm. These findings provide insight into how the fast-growing S. mutans creates nutrient-depleted regions that affect the survival and metabolic behavior of other bacterial species within oral biofilm.