The invention discloses a guide positioning wheel set of a piston rod of a hydraulic seed cutting machine. The guide positioning wheel set comprises a mounting plate; the mounting plate is provided with a limiting groove, multiple connecting holes and multiple mounting holes; the limiting groove is arranged at the upper part of the mounting plate; the multiple connecting holes are formed in the mounting plate; the limiting groove communicates with the multiple connecting holes; the mounting plate is connected with the hydraulic seed cutting machine through the multiple mounting holes; the upper part of a connecting shaft is mounted in the connecting holes; a positioning hole is formed in the upper part of the connecting shaft, and is corresponding to the limiting groove; a fixed rod is mounted in the limiting groove, and can penetrate through the positioning hole; and a guide limiting wheel is movably mounted at the lower part of the connecting shaft. The guide positioning wheel set is connected with the piston rod of the hydraulic seed cutting machine; and when the piston rod is suffered from the external force effect to generate a torque for rotating tendency, the guide limiting wheel and the connecting shaft can generate reactive force to prevent rotation of the piston rod, so that the stability of the piston rod is improved.