<p>Medical studies have shown that vitamin D deficiency is strongly associated with several metabolic disorders, including diabetes, cardiovascular diseases, and cancer. It is crucial to regularly check the concentration of vitamin D in the blood serum. Traditional methods for detecting 25-hydroxyvitamin D₃ [25(OH)D₃] as a marker of vitamin D status are expensive and time-consuming, and require a skilled workforce and specialized laboratory. This study developed a simple and cost-effective fluorescence system for 25-hydroxyvitamin D₃ determination. The fluorescent APTA-nanobiosensors were fabricated using cadmium telluride quantum dots modified with thioglycolic acid (CdTe-TGA QDs) and functionalized with thiol-25(OH)D₃-aptamer through ligand exchange. The thiol-25(OH)D₃-aptamer interacted directly with CdTe-TGA QDs, increasing fluorescence intensity. However, it decreased when the target molecules of 25-hydroxyvitamin D₃ were introduced. The structural and morphological characteristics of APTA-nanobiosensors were confirmed by UV–visible spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), x-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FESEM), energy dispersive x-ray spectroscopy (EDX), transmission electron microscopy (TEM), and dynamic light scattering (DLS). According to the typical Stern–Volmer equation, the relationship between fluorescent quenching and target concentration was linear with a detection limit of 1.35 × 10^–8 M, a quantification limit of 4.50 × 10^–8 M, and a relative standard deviation of 1.75%. The optimized APTA-nanobiosensor demonstrated high specificity toward the target and stability over 28 days. Furthermore, it detected 25-hydroxyvitamin D₃ in human serum and urine with a recovery rate of 96.00–101.14%. The results indicate that the APTA-nanobiosensors could be valuable in developing robust sensing technology for low-concentrated analytes.</p> Graphical Abstract <p></p>