<p>The design and fabrication of fluorescent nano-biosensors for detecting various analytes in complex matrices are of great interest to researchers. One of the interests of researchers in the medical field is the sensitive detection and measurement of glucose in biological fluids, as diabete is a chronic metabolic disease with a high risk of death in the world. In this work, a fluorescent nano-biosensor, based on modified cadmium telluride quantum dots capped with thioglycolic acid) CdTe/TGA QDs (with poly (acrylic acid)-co-(vinyl phenyl boronic acid) grafted onto gelatin, (CdTe-(PAA-<i>co</i>-VPB)-<i>g</i>-GE QDs), was fabricated for the detection of glucose in biological fluids. One of the components in the modified biopolymer coating is VPB, because it tends to cis-diol interactions with glucose. Therefore, the selectivity and sensitivity of the nano-biosensor are significantly increased. Then, its synthesis method was optimized for high fluorescence intensity with high sensitivity and stability using a 4-factor Box–Behnken statistical design. By adding different amounts of glucose to the nano-biosensor, its fluorescence emission is linearly quenched by a possible mechanism of Photo induced Electron Transfer )PET (. Also, the optimized nano-biosensor has very good repeatability and selectivity with a stability of 120 days. The detection limit of glucose is 20.26 ± 1.2 × 10^− 9 mol L^− 1, with a dynamic range from 2.26 ± 1.22 × 10^− 7 to 1.11 ± 1.14 × 10^− 3 mol L^− 1. In addition, the prepared nano-biosensor is capable of measuring glucose in biological fluids with satisfactory results.</p>