The invention provides a positioning tool of an eccentric shaft and a machine tool. The positioning tool comprises a chuck assembly, a transmission part eccentrically fixed to the chuck assembly, an angle positioning jacket, a front tip, and a rear tip, the angle positioning jacket is connected with the transmission part and is provided with a positioning hole, the positioning hole is conical, andis used for radially fixingthe eccentric shaft, the front tip is fixedly arranged on the chuck assembly, the rear tip is fixedly arranged on the machine tool, and the front tip and the rear tip are used for abutting against the two axial ends of the eccentric shaft. Through the technical scheme, different specifications of eccentric shafts can be rapidly clamped and positioned, the machining precision of the eccentric shaft can be improved, the scrappage is reduced, the positioning tool is simple in structure, only few parts like a spline positioning sleeve need to be replaced, the replacingwork intensity of the positioning tool of the eccentric shaft can be reduced, and the replacing efficiency of the positioning tool is improved.