The invention provides high-strength alloy structural steel for a hydraulic cylinder of a fracturing pump. The steel comprises the following chemical elements in percentage by weight: 0.30-0.36% of C,0.23-0.28% of Mn, less than or equal to 0.015% of P, less than or equal to 0.010% of S, 0.05-0.10% of Si, 3.20-3.30% of Ni, 1.40-1.50% of Cr, 0.55-0.60% of Mo, 0.11-0.15% of V, less than or equal to1.5 ppm of H, less than or equal to 112 ppm of O, less than or equal to 70-120 ppm of N and the balance Fe. Meanwhile, the invention further provides a preparation method of the structural steel, thestructural steel obtained through the preparation method is used for manufacturing a hydraulic cylinder of a fracturing pump, under the severe working condition of shale gas exploitation, the servicelife of the product can be prolonged, the use cost can be reduced, the quality is stable and reliable, unpredictable cracking failure and production accidents in the operation process are avoided, andthe requirements of oil-gas field deep well fracturing and shale gas exploitation are met.