The invention relates to a method for sheeting peanut root tip cell chromosome in the mitosis metaphase, which comprises the following steps that: a peanut root tip is selected, and paradichlorobenzene is utilized for soaking pretreatment; the root tip is put into carnoy stationary liquid for room temperature treatment; acetic acid is used for soaking, the root tip of a meristematic zone is cut and put on a glass slide, the center of a cover glass is tapped to disperse the cells, a cell wall is heated by fire for dissociating and softening, and a filter paper is used for removing the excess acetic acid; the cell which is in the mitosis metaphase and has smoothly dispersed chromosome is sheeted and refrigerated, the cover glass is removed, and the glass slide is dehydrated; and stain zoning or fluorescence in-situ hybridization are carried out. The obtained sheet has leveled cells and dispersed chromosome, and more cells are in the metaphase; and the acetic acid medium is used for direct compression, the reagent has small influence to the chromosome, so the method is easy to operate and control, has simple steps and high sheeting efficiency. The method is suitable to not only peanuts but also sesame, paddy, corns, soybeans, green beans or tomatoes and other various plants, and has good operability.