As glucose biosensors play an important role in glycemic control, which can prevent the diabetic complications, the development of a glucose sensing platform is still in needed. Herein, the first proposal on the in-house fabricated paper-based screen-printed ionic liquid/graphene electrode (SPIL-GE) modified with MXene (Ti₃C₂T_x), prussian blue (PB), glucose oxidase (GOx), and Nafion is reported. The concentration of PB/Ti₃C₂T_x was optimized and the optimal detection potential of PB/Ti₃C₂T_x/GOx/Nafion/SPIL-GE is −0.05 V. The performance of PB/Ti₃C₂T_x/GOx/Nafion modified SPIL-GE was characterized by cyclic voltammetry and chronoamperometry technique. This paper-based platform integrated with nanomaterial composites were realized for glucose in the range of 0.0–15.0 mM with the correlation coefficient R² = 0.9937. The limit of detection method and limit of quantification were 24.5 μM and 81.7 μM, respectively. In the method comparison, this PB/Ti₃C₂T_x/GOx/Nafion/SPIL-GE exhibits a good correlation with the reference hexokinase method. This novel glucose sensing platform can potentially be used for the good practice to enhance the sensitivity and open the opportunity to develop paper-based electroanalytical devices.