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The development of a facile, reductant- and stabilizer-free alkali-mediated synthesis of gold nanoparticles (AuNPs) within cotton fibers for biocompatible textiles is described in this study. Uses of external chemicals compromises the biocompatibility of AuNPs. Additionally, alkali-mediated synthesis of AuNPs inside cotton has not been successful previously. Herein, AuNPs are synthesized in situ within the interior of cotton fibers under alkaline conditions using a Au(III) (Au3+) precursor (tetrachloroauric acid, HAuCl4) without adding any reducing or capping agents. A mechanism of AuNP formation using cotton cellulose has been studied to understand the formation of Au3+-cellulose complexes and their subsequent reduction to zerovalent atoms. AuNP-infused cotton fibers (AuNP-cotton) were analyzed using ultraviolet–visible (UV–vis) spectroscopy, and the surface plasmon resonance (SPR) peak at 538 nm confirms the reduction of the Au3+ precursor and the formation of metallic AuNP (Au0). The obtained AuNPs are spherical with an average diameter of 12.25 nm. Au content in treated cotton was determined to be 1.66 wt % using graphite furnace atomic absorption spectroscopy (GFAAS). High-resolution electron microscopy techniques reveal the internal formation of AuNPs within the cotton fibers, which are uniformly distributed without agglomeration. The AuNPs incorporated into the cotton fabric demonstrated superior laundering durability under intense mechanical force, retaining 82% AuNPs after 50 home laundering cycles. Rapid and efficient catalytic activity of AuNP-cotton demonstrates easy accessibility of the AuNP surface for chemical interactions across multiple cycles. Notably, nontoxicity toward mouse fibroblast cells and efficient photothermal conversion efficiency (2.3–6.4%) suggest that AuNP-cotton has strong potential to support human cell growth and suitability for biocompatible applications.