In this paper, a potentiometric method is used for monitoring the concentration of glutamine in the bioprocess by employing silicon nanowire biosensors. Just one hydrolyzation reaction was used, which is much more convenient compared with the two-stage reactions in the published papers. For the silicon nanowire biosensor, the Al2O3 sensing layer provides a highly sensitive to solution-pH, which has near-Nernstian sensitivity. The sensitive region to detect glutamine is from ≤40 μM to 20 mM. The Sigmoidal function was used to model the pH-signal variation versus the glutamine concentration. Compared with the amperometric methods, a consistent result from different devices could be directly obtained. It is a fast and direct method achieved with our real-time setup. Also, it is a label-free method because just the pH variation of the solution is monitored. The obtained results show the feasibility of the potentiometric method for monitoring the glutamine concentrations in fermentation processes. Our approach in this paper can be applied to various analytes.