This research investigated optimization of sterilization techniques and cytokinin concentrations to improve the in vitro propagation of turmeric (Curcuma longa). Given the limitations of conventional cultivation methods, such as susceptibility to diseases, slow growth and more area requirements, tissue culture techniques provide a more efficient alternative for controlling propagation. The study conducted in the Tissue Culture Laboratory' Faculty of Environmental Agricultural Sciences, Arish University, Egypt. During the initial establishment phase, rhizome buds were sterilized using commercial Clorox at concentrations of 10%, 15%, and 20% for varying periods (5, 10 and 15 min.). The most successful sterilization approach was achieved with 20% Clorox applied for 10 minutes, resulting in a 90% survival rate and minimal contamination. For shoot multiplication, different cytokinins such as thidiazuron (TDZ), benzyl adenine benzyl adenine (BA), and Kinetin were evaluated separately at different concentrations. The best shoot proliferation was observed with 1.0 mg l⁻¹ TDZ. In the rooting phase, growth media fortified with Naphthalene Acetic Acid (NAA) at concentrations ranging from 0.5 to 2.0 mg l⁻¹ significantly influenced the root growth' Moreover, rooting medium supplemented with 1.5 mg l⁻¹ NAA produced the longest roots. For acclimatization, a mixture of peat moss, vermiculite, and washed sand in a 1:1:1 ratio (V/V/V) was used, resulting in an 80% survival rate for the plants. The findings highlight the importance of optimized sterilization methods and growth regulator concentrations in improving the tissue culture propagation of turmeric, offering promising potential for the large-scale production of this medicinally important plant.