The invention discloses theta 28-36mm HRB600 high-strength anti-seismic reinforcement steel bars for a hydropower project. The reinforcement steel bars are characterized in that steel comprises the following chemical components by weight: C 0.23-0.26wt%, Si 0.50-0.65wt%, Mn 1.45-1.55wt%, S 0.025-0.035wt%, P 0.030-0.040wt%, Cr 0.12-0.18wt%, Mo 0.020-0.035wt%, V 0.125-0.140wt%, and the balance beingFe and inevitable impurities. The invention also discloses a preparation method of the large-specification HRB600 high-strength anti-seismic reinforcement steel bars. A vanadium, chromium and molybdenum composite microalloying process is adopted, microalloying strengthening elements such as vanadium-nitrogen alloy, ferrochromium and ferromolybdenum are added into the steel, and the low final rolling temperature is controlled by a pre-penetrating water-controlled cooling device (4.9m in length) in the steel rolling process, the formation and precipitation of fine and dispersed microalloy carbon (nitrogen) secondary phase are promoted, and the toughness is obviously improved during improvement of steel strength. The reinforcement steel bars can be used for sdam and flood control tunnel construction engineering and road and bridge engineering of the Jinshajiang hydropower station, and has the advantages of low production cost, strong process applicability, reliable operation, stable product and the like.