Funding information Higher Education Commission of PakistanNanoparticles (NPs) are the fundamental backbone of nanotechnology due to their wide biomedical applications. Biogenic synthesis of gold NPs (AuNPs) using plant extracts are beneficial due to their nontoxicity, eco-friendliness, and simplicity. In this study, the HE-AuNPs were synthesized using Heliotropium eichwaldi L (HE) extract and then characterized by UV-visible (UV-vis) spectroscopy, Fourier transform-infrared (FT-IR), scanning electron microscope (SEM), X-ray diffractometer (XRD), and electron diffraction X-ray spectroscopy (EDX) analysis. UV-vis spectroscopy revealed a sharp peak λ max 567 nm, FT-IR analysis confirmed the existence of different coating and reducing organic molecules, SEM analysis showed the elongated rod-shaped morphologies with 20 nm size, XRD analysis authorized the crystalline structure of AuNPs with an average size of 11.11 nm for four key peaks (38.31 , 44.39 , 64.76 , and 77.65 ), and EDX analysis revealed that Au (44.33%) was the main element of AuNPs compared to other surface coated elements C (35.05%) and O (20.62%). The influence of various intrinsic factors like pH, temperature, metal salt, HE extract, contact time, and stability period was also studied by UV-vis spectroscopy. Moreover, the AuNps were evaluated for their anticholinesterase (anti-AChE) potential to treat Alzheimer's disease (AD). HE-AuNPs exhibited superb anti-AChE activity (67 ± 0.03%) with 88 ± 0.31 μg IC 50 , compared to pure Au salt, which showed 7 ± 0.22% inhibition. According to the Lineweaver-Burk plot, HE-AuNPs inhibit AChE noncompetitively, that is, K m remains unchanged, whereas V max decreased from 20 to 6.24 (42.4% to 82%). HE-AuNPs do not affect K Iapp , whereas increased V maxiapp from 17.5 to 29 (13% to 86.4%). The K m (Michaelis-Menten kinetic constants), K I (dissociation constant), and K i (inhibitory constant) were found to be 0.041 mM, 66 μg, and 25 μg, respectively. Overall, our results highlighted the HE-AuNPs promising latent as anti-AChE, and the green methodology allows us to suggest these NPs as suitable agents for the development of drugs helpful in the treatment of AD.