Objective To design and fabricate a foldable microplate for single cell culture, and establish the finite element model of the folding microplate to calculate the traction force of single cell when it contracted in three-dimensional state. Methods Based on the experimental data of single cell cultured on the foldable microplate, the folding angle of the microplate was calculated. The finite element simulation was used to calculate the bending moment of the microplate, and the function relation between bending moment and folding angle M=M(θ) was obtained. Then the relation between traction force and bending moment F= F (M) was derived. Results The folding angle of the microplate with HSF and MC3T3-E1 were determined among the range of 73°~173° and 49°~138°, respectively. The single-cell tractive forces of HSF and MC3T3-E1 were calculated in the range of 0-250 nN and 50-150 nN, respectively. Conclusions A new method for measuring the traction force of a single cell in a three-dimensional structure is proposed. This method provide a new insight for further development of the calculation of the traction force of cells in the three-dimensional physiological-related adhesion, spreading and migration behavior.