Cassia grandis (CG) fruit extract, traditionally used in folk medicine, has found a new role as an eco-friendly material for synthesizing and stabilizing biogenic AuNPs, particularly for glucose and vitamin C detection. Based on the phenomenon related to Rayleigh scattering, the synthesis conditions have been optimized at a temperature of 100 °C for 50 min with 0.5 mM of Au3+ in an extract ratio of 1:5. The formation mechanism and catalytic mechanism of the biogenic AuNPs in the TMB oxidation is also elucidated in detail. The morphological properties of the synthesized AuNPs were investigated using FT-IR, XRD, SEM-EDS, TEM, and DLS. The AuNPs are approximately 16.95 ± 2.57 nm in diameter, exhibiting a spherical structure. Notably, the AuNPs synthesized and stabilized by CG-extract phytochemicals demonstrate a zeta potential of −40.33 mV, surpassing that of nanoparticles derived from several other natural extracts. Even after one month of storage, this value decreased by approximately 7.5 %. Moreover, under the established optimal conditions, the colorimetric assay using CG-AuNPs showed remarkable sensitivity and selectivity for both glucose and vitamin C, with LODs of 8.68 and 5.26 μM in linear ranges of 10–800 and 5–50 μM, respectively.