The invention discloses a preparation method of a fruit and vegetable fresh-keeping agent. The method comprises the following steps: firstly, taking coptis chinensis, pyrola rotundifolia, rheum officinale, polygonum cuspidatum, gallnut, chuanxiong rhizomes and scutellaria baicalensis, and uniformly mixing the coptis chinensis, the pyrola rotundifolia, the rheum officinale, the polygonum cuspidatum, the gallnut, the chuanxiong rhizomes and the scutellaria baicalensis which are taken; performing extraction by a supercritical carbon dioxide fluid extraction method, and then performing separation so as to obtain an extracting solution; taking syzygium aromaticum, cinnamon, fructus forsythiae, radix stemonae and polygonum multiflorum, adding water to the syzygium aromaticum, the cinnamon, the fructus forsythiae, the radix stemonae and the polygonum multiflorum which are taken, to perform decocting so as to obtain a decoction solution, filtering the decoction solution, concentrating the filtered solution under a reduced pressure, and carrying out cooling naturally, thereby obtaining the concentrated solution; and mixing the obtained extracting solution with the concentrated solution so as to obtain the fruit and vegetable fresh-keeping agent. The fruit and vegetable fresh-keeping agent prepared by the method disclosed by the invention can effectively inhibit diseases caused by pathogenic bacteria, of picked fruits and vegetables, can significantly reduce loss caused by the rot of picked fruits and vegetables, and can improve the quality of picked fruits and vegetables; in addition, the fruit and vegetable fresh-keeping agent is higher in food safety, does not generate pollution to the environment, and can effectively reduce the usage and the residue of synthetic bactericides and chemical pesticides.