The development of innovative, eco-friendly methods for synthesizing functional nanoparticles is crucial in advancing cancer therapeutics. This study highlights a one-pot in situ synthesis of paclitaxel-functionalized gold nanoparticles (PTX-AuNPs), with paclitaxel serving as both the reducing and stabilizing agent. The synthesis process was validated using UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and high-resolution transmission electron microscopy (FEG-TEM). High-performance liquid chromatography (HPLC) confirmed the purity and structural integrity of paclitaxel before and after synthesis. The resulting PTX-AuNPs exhibited potent anticancer activity against human cervical cancer (SiHa) and human colon cancer (HT-29) cell lines, with a significantly stronger effect on the HT-29 cell line. A concentration-dependent reduction in HT-29 cell growth was observed as nanoparticle concentrations increased from 10 µg/mL-20 µg/mL. Molecular docking studies further demonstrated paclitaxel's strong binding affinity (-8.5 kcal/mol) to β-Tubulin, elucidating its anticancer mechanism. This cost-effective and environmentally friendly approach offers significant promise for enhancing cancer treatment strategies.