Abstract: The study systematically optimized the protoplast fusion system induced by the PEG-high Ca²⁺ high pH method using the leaves and petals of Dahlia imperialis and‘Caf é au Lait Royal’ as test materials. The results showed that when the protoplast density was adjusted to 3.0×10⁵ protoplasts/mL, the fusion rate of leaf and petal protoplasts could reach 15.57% when they were treated with 30% PEG-4000 and 20% PEG-6000, respectively, for 25 min, then added with a high Ca²⁺-high pH solution and left standing at room temperature for 10 min. PEG fusion by protoplasts from different sources showed that the fusion rate of dahlia protoplasts could reach 5.70%. This study shows for the first time that the protoplasts from different tissue sources of dahlia have specific responses to the molecular weight and concentration of PEG, and the established differential parameter system can provide technical support for somatic cell hybrid breeding of self-pollinated crops.