The invention discloses a preparation method of a Calibrachoa protoplast. The method comprises the following steps: preprocessing tender leaves in 3-5DEG C clear water under dark conditions for 20-25h; carrying out enzymatic hydrolysis on the preprocessed blades by using an enzymatic hydrolysis solution; adding a CPW washing solution having a same volume with the enzymatic hydrolysis solution, shaking up, filtering by using a 300 mesh cell sieve, centrifuging at a rotating speed of 1100r/min for 2min, and absorbing the obtained supernatant; and adding a CPW washing solution to the obtained precipitate to wash a protoplast, centrifuging at a rotating speed of 1100r/min for 2min, collecting the obtained precipitate, adding a suspension, and suspending to obtain the protoplast with the output of (4.7-5.3)*10<6>/g and the activity of 85%, wherein the activity of 80% of the protoplast is maintained 9h later. The protoplast with high activity, high output and good state is obtained through a separation technology, protoplast culturing and plant regeneration conditions are met, and an experiment material is provided for plant protoplast fusion cultivation of new kinds.