This study aimed to establish and optimize an open in-bottle micro-cutting technique for Taxus × media. An orthogonal experimental design was adopted. Four core factors were optimized: light regime, plant growth regulator (PGR) type, PGR concentration, and PGR soaking time. Analysis of variance (ANOVA) and Duncan’s multiple-range test were performed to analyze the statistical significance of each factor. The optimal combination was identified. Branches were soaked in a 300 mg·L⁻¹ IBA solution for 15 min. They were then cultured under 12 h alternating light/dark conditions in an illumination incubator. Under these conditions, the cutting rooting rate reached a maximum of 45.83%. The optimized open tissue culture system established in this study provides reliable technical support for the clonal propagation of T. × media.