The invention discloses a pinus massoniana needle protoplast separation purification and instantaneous efficient conversion method. Firstly, a combinative enzyme is used for conducting enzymolysis onpinus massoniana needle cells to form protoplast, after the obtained protoplast is subjected to separation, purification and pre-treatment, a PEG method is used for transferring an empty vector carrying a GFP tag into the pinus massoniana protoplast, and through culture, the empty vector is expressed in the protoplast. According to the pinus massoniana needle protoplast separation purification andinstantaneous efficient conversion method, a pinus massoniana protoplast instantaneous expression system is built, operation and implementation are easy, the conversion efficiency of the method can reach 60%, and a fast convenient studying platform is provided for pinus massoniana functional gene studying; the enzymolysis system used in the pinus massoniana needle protoplast separation purification and instantaneous efficient conversion method is suitable for pinus massoniana seedlings, by means of young tender needles, the high-quality protoplast can be obtained, the activity of the protoplast can reach 80% or above, and the method is also suitable for wild mature needle tissues.