Prunus xueluoensis, a unique Prunus germplasm resource native to China, exhibits significant ornamental value due to its short juvenile phase, early flowering period, abundant flowers, and elegant tree form. However, the lack of an efficient regeneration and genetic transformation system has hindered its genetic improvement and wider application. In this study, we focused on optimizing the tissue culture conditions for P. xueluoensis and establishing an Agrobacterium-mediated transient genetic transformation system. We first determined the optimal medium compositions for different stages of tissue culture, including seed germination, callus induction, adventitious bud differentiation, and rooting. For seed germination, the optimal medium was MS supplemented with 200 mg/L GA3 and 4 mg/L 6-BA. For callus induction, the best medium was MS containing 2.00 mg/L 6-BA, 1.00 mg/L NAA, and 200 mg/L VC. Adventitious bud differentiation was favored on MS medium with 1.00 mg/L 6-BA, 0.10 mg/L NAA, and 200 mg/L VC, while rooting was optimal on 3/4 MS medium supplemented with 0.50 mg/L NAA. Subsequently, we established an Agrobacterium-mediated transient genetic transformation system using stem segments of P. xueluoensis as explants. Through orthogonal experiments, we identified the optimal conditions for genetic transformation as pre-cultivation for 2 days, an Agrobacterium concentration of OD₆₀₀ = 0.6, an infection time of 30 min, and co-cultivation for 3 days. Under these conditions, the transient genetic transformation efficiency reached 10.42%, as confirmed by PCR and GFP fluorescence detection. This study provides a reliable transient genetic transformation system for P. xueluoensis, facilitating further functional gene analysis and genetic improvement of this valuable ornamental species.