Functionalized porous nanomaterials have been introduced as novel substrates with high surface area and large porosity for the construction of efficient catalysts in sensors. In this regard, nano X zeolite (NX), as a kind of aluminosilicate framework material, can be used as a hosting material for nanocatalysts in the design and fabrication of electrochemical sensors. Herein, a sensor based on multi-walled carbon nanotube (MWCNT) and NX material as a substrate for AuNi bimetallic nanoparticles obtained from agro-waste stem sweep was fabricated to detect nanomolar concenteration of glucose. The catalyst has provided a desired analytical performance for the glucose sensor with low limit of detection (0.063 μM) in the linear range of 1–1900 μM and sensitivity of 662.93 μA mM−1. The results depend on the presence of NX in the sensor and confirm the preference of a porous host existence for the AuNi catalyst in the sensing process. In AuNi/NX/MWCNT electrocatalyst, the disaggregation of Au nanoparticles by decorating on sacrificial Ni and the creation of abundant active sites through NX make the acceptable results for non-enzymatic detection of glucose in the human serum samples.