Quercus suber L. (Q. suber) is an evergreen tree species known for producing high-quality cork. Traditional seed propagation of Q. suber has low viability and is time-consuming. Therefore, we used young stem segments of 2~3-year-old seedlings as explants, and optimized protocols for tissue culture and rapid micropropagation of Q. suber. The best disinfection method was 0.10% HgCl2 (v/v) for 5 min. 0.50 g·L−1 Poly Vinyl Pyrrolidone (PVP) is the best anti-browning agent with a significant reduction in browning by nearly 1.76-fold (58.89% → 33.33%). Woody Plant Medium supplemented with micronutrients and vitamins from Murashige and Skoog Medium (WPMS) was found to be the most suitable for shoot formation. The optimal hormone ratio for development of shoots from axillary buds was 0.60 mg·L−1 6-benzyladenine (6-BA). Among the cytokinins tested, 0.50 mg·L−1 6-BA was the most suitable for development of shoots from axillary buds. In additon, the highest percentage of rooting explants (66.67%) and rooting number (3.03) was obtained on WPM basal medium supplemented 0.20 mg·L−1 IBA and 0.20 mg·L−1 NAA. In summary, we have established a set of protocols for tissue culture and rapid micropropagation of Q. suber. These findings lay the foundation for rapid micropropagation and genetic improvement.