The invention discloses a preparation method of a thalassia hemperichii protoplast and belongs to the field of plant cell engineering. The preparation method comprises the following steps: S1, takingyoung leaves of thalassia hemperichii, washing them with sterile seawater, and temporarily culturing them to obtain a raw material 1; S2, putting the raw material 1 obtained in the step S1 into a pretreatment liquid for soaking to obtain a raw material 2; S3, taking out the raw material 2 obtained in the step S2, sucking dry water, cutting them into blocks, adding enzyme liquid for enzymolysis, and obtaining enzymolysis tissues; S4, filtering the enzymolysis tissue obtained in the step S3 to obtain filtrate; S5, centrifuging the filtrate obtained in the step S4, discarding the supernatant, suspending the precipitate with a resuspension solution, centrifuging, and discarding the supernatant to obtain a precipitate; S6, suspending the precipitate obtained in S5 with a resuspension solution to obtain the thalassia hemperichii protoplast. The preparation method of the thalassia hemperichii protoplast is established for the first time, and the thalassia hemperichii protoplast obtained by the method is high in yield and high in survival rate.