Genetic transformation is an important method for molecular breeding and gene function research. To establish an efficient genetic transformation system for "Jingzao 39" jujube, leaves of "Jingzao 39" were utilized as explants for genetic transformation. This study investigated the effects of kanamycin (Kan) selection pressure, concentrations of cefotaxime (Cef) and timentin (Tim), pre-culture duration, various bacterial suspension densities, infection duration, acetylsyringone (AS) concentration, and co-cultivation time on the transformation. The results showed that the optimal genetic transformation system for "Jingzao 39" leaves consisted of a Kan concentration of 5 mg/L, Cef concentration of 50 mg/L, Tim concentration of 250 mg/L, a pre-culture period of 4 days, bacterial suspension with an OD600 value of 0.6, infection duration of 20 minutes, AS concentration of 200 μmol/L, and co-cultivation for 4 days. Both fluorescent lights detection and PCR analysis confirmed the integration of the exogenous gene into the "Jingzao 39" genome, indicating successful Agrobacterium-mediated genetic transformation of "Jingzao 39" leaves. This study will provide important support for gene functional analysis and molecular breeding in jujube.