Abstract: To screen the optimal medium formulation for callus induction from leaves of Camellia fascicularis, leaves from tissue-cultured seedlings were used as materials. The effects of hormones, light conditions, explant orientation, and medium composition on leaf callus induction were investigated, and the callus induction medium formulation was optimized through orthogonal experiments. The results showed that placing the leaf abaxial side in contact with the medium and maintaining complete darkness during cultivation were most conducive to callus induction. The optimal medium formulation for leaf callus induction was 1/2MS + 45 g·L⁻¹ sucrose + 3.0 mg·L⁻¹ NAA. This study established a technical system for callus induction from leaves of C. fascicularis, laying a preliminary foundation for the subsequent development of a genetic transformation system using callus.