The invention discloses a preparation method and an application of a brassica napus BnPABP5-3 promoter (PBnPABP5-3). A genome walking method is utilized to clone a PBnPABP5-3 sequence: an SDS (sodium dodecyl sulphate) cracking process is applied to extract genome DNA; in different systems, endonucleases DraI, EcoRV, PvuII and StuI are used to digest DNA, and the digested DNA is respectively connected with joint segments, then primary PCR (Polymerase Chain Reaction) amplification is carried out by taking the DNA connected with the joint segments as templates, and secondary PCR amplification is carried out by taking 50-time diluted solution of products of the primary PCR as a template, thus the PBnPABP5-3 is obtained. The PBnPABP5-3 can be applied to the anther, pollen grain, root tip and ovule of a plant. The promoter has the function of driving expression of a foreign gene, the expression part is in the anther, pollen grain, root tip and ovule of the plant, and the promoter has application potential in the improvement of the safety of rape edible oil, the improvement of the stress resistance and lodging resistance, improvement of the quality of crops, artificial sterile line creation, germ plasma resource enrichment and the like.