The invention discloses a preparation method of high-quality 40Cr alloy structure straight-bar round steel. The preparation method includes the following steps, namely, the step A of molten iron pretreatment and desulfuration, the step B of molten iron smelting, the step C of deoxidization alloying, the step D of molten iron LF furnace refining, the step E of molten iron VD furnace vacuum refining, the step F of molten iron casting, the step G of steel billet heating, the step H of rolling and the step I of steel finishing. The high-quality 40Cr alloy structure straight-bar round steel produced through the method is high in cleanliness and low in inclusion content and content of harmful elements P and S, has high strength, excellent ductility and toughness, excellent impact toughness and excellent upsetting deformation capacity, and can well meet the requirements of the field of high-end machine manufacturing industries.