<p><i>Cannabis</i> <i>sativa</i> L., a versatile plant with medicinal, industrial, and psychoactive uses, widely recognized for its psychoactive compound THC (Δ9-tetrahydrocannabinol), also contains over 104 cannabinoids, such as cannabidiol (CBD), used to treat Parkinson’s disease, epilepsy, multiple sclerosis, and anxiety disorders. This study explores the impact of green synthesized sulphur nanoparticles (gSNPs) on <i>in vitro</i> regeneration of <i>C. sativa</i>. ½ Murashige and Skoog (MS) medium was used to germinate surface-sterilized seeds. Axillary buds were collected from <i>in vitro</i> germinated seeds in order to induce shoots in MS media supplemented with 0.5, 0.25, 2.5, and 5.0 µM of benzylaminopurine (BAP), thidiazuron (TDZ), and kinetin (KIN). It was observed that the most effective supplementation for both shoot induction and proliferation was 2.5 µM TDZ and 5.0 µM gibberellic acid (GA₃) in MS medium. So, this combination was taken for studying effect of up to 60 mg L⁻¹ gSNPs. Out of 50 axillary buds inoculated, approximately 40 buds showed shoot induction and proliferation on MS medium supplemented with 2.5 µM TDZ, 5.0 µM GA₃, and 50.0 mg L⁻¹ gSNPs, significantly outperforming the control without gSNPs. Rooting experiments with 50.0 mg L⁻¹ gSNPs in ½ MS medium with up to 5.0 µM NAA and IBA revealed that 2.5 µM indole- 3-butyric acid (IBA) with 50.0 mg L⁻¹ gSNPs yielded highest number of roots (17.7 ± 1.20) and root length (12.0 ± 1.52 cm). Genetic fidelity analysis was performed using six Start Codon Targeted (SCoT) DNA primers. Five primers yielded results and showed no polymorphism between gSNP-treated, untreated, <i>in vitro</i>-grown, and greenhouse acclimatized plants, confirming the genetic stability of the micropropagated plantlets. This standardized protocol presents a novel approach to <i>C. sativa</i> micropropagation using gSNPs with potential applications in both research and industrial applications of <i>Cannabis</i>.</p>