The invention provides an immunofluorescence method for locating a woody plant pollen tube protein and a kit thereof. The kit includes a fixing solution, an enzymolysis solution, a blocking solution,a primary antibody and a secondary antibody; the fixing solution contains PEPES, sucrose, Triton X-100, paraformaldehyde, NaCl, KCl, Na2HPO4 and KH2PO4, the enzymolysis solution contains cellulase, macerozyme, NaCl, KCl, Na2HPO4 and KH2PO4, the blocking solution contains bovine serum albumin, NaCl, KCl, Na2HPO4 and KH2PO4, the primary antibody is a target protein antibody, and the secondary antibody is a FITC-labeled goat anti-rabbit secondary antibody. The method adopts an apple pollen tube as a test material, after enzymatic hydrolysis of the wall of the apple pollen tube, immunofluorescentlabeling is performed. The method can directly, efficiently, sensitively and specifically locate the functional protein in the pollen tube, and provides a technical system for visually identifying theexpression of the functional protein in the pollen tube of a woody plant and locating the functional protein in the pollen tube of the woody plant.