The invention discloses a preparation method of 2,3,5,6-tetra-substituted symmetric pyridine. Specifically, the symmetric 2,3,5,6-tetra-substituted pyridine is prepared by taking beta-ketonic ester as a reaction substrate, a metal copper compound as a catalyst, a peroxide reagent as an oxidizing agent, inorganic ammonium salt as a nitrogen source and N-methylformamides as an organic solvent (also as a carbon source) and through carbon-hydrogen and carbon-nitrogen removal, dehydration condensation and an oxidation aromatization reaction. The preparation method disclosed by the invention has the characteristics of easily available raw materials, cheap and low-toxic catalyst, high catalytic efficiency, wide substrate range, simple and convenient operation, greenness and environmental friendliness and the like.