Abstract: For the disadvantages of the six-axis force sensor based on the classical Stewart structure, a double-layer prestressed six-axis force sensor is designed and manufactured, and accuracy analysis and experimental research are carried out. Firstly, structure characteristics of the six-axis force sensor are introduced. The mathematical model of the sensor and the representation of the pre-tightening force are established by using screw theory. In order to improve the measurement accuracy, a method for reducing the error of the pre-tightening branch's structural deformation by increasing the pre-tightening force is proposed on the basis of analyzing the structure of pre-tightening branch. Secondly, the structural deformation of pre-tightening branch under different pre-tightening forces is simulated by FEM (finite element method). Finally, the sensor prototype is manufactured and the calibration system is developed. The calibration experiment is performed under different pre-tightening forces. It is shown that the maximum Error I and Error II are reduced from 2.73%, 2.43% to 0.41%, 0.64% of the full scales respectively by increasing the pre-tightening force. The experiment results indicate that the error caused by the pre-tightening branch's structural deformation is reduced effectively and the measuring accuracy of the sensor is improved by increasing the pre-tightening force, which verifies the correctness of the theoretical analysis and simulation.