Establishing a transformation system is fundamental to transgenic study. We developed an efficient Agrobacterium tumefaciens-mediated transformation and screening system for Cucurbita moschata ‘Jinhui 1’. The leaves from tissue culture were excellent explants for transformation. To construct a genetic transformation system, factors affecting the transformation frequency included pre-culture time of explants, infection time, and concentration of acetosyringone (AS). Additionally, co-culture time and the concentration of antibiotics (Carb, Cefotaxime and Kanamycin), which inhibit the growth of Agrobacterium, were also examined in the adventitious bud. The data were analyzed by DPS software, and a suitable genetic transformation system was established. Pre-culturation for 0 days, infection for 30 min, AS at 100 mg·L^–1 and co-culture for 5 days produced the highest transformation efficiency. The medium with Cefotaxime (500 mg·L^–1) and Kanamycin (100 mg·L^–1) was optimal for selecting transgenic plant cultivation-resistant shoots on MS for eventual root induction. PCR and Southern blot results showed the Kanamycin-resistant exogenous gene had been integrated into the genome of Cucurbita moschata.