Objective The value of Poisson"s Ratio will affect the measurement accuracy of the elastic modulus of cells, and the accurate Poisson"s Ratio is helpful to master the mechanical properties of cells. Methods Studies on the mechanical properties of cells usually assume that the cells are incompressible, that is, the Poisson"s ratio is 0.5, but in fact, the Poisson"s ratio of different cells is different. In this paper, a method for the determination of Poisson"s ratio basedon Micropipette aspiration technique is proposed. Based on the assumption of deformation symmetry, the analytical expression between Poisson"s ratio and the amount of deformation was derived by extracting the extrusion deformation characteristics of the cells under Micropipette aspiration according to the generalized Hooke"s law. The accurate determination of Poisson"s ratio of cells was realized according to the position changes of the markers on the surface of the cell membrane. Results The Poisson"s ratio of LNCaP cells in prostate cancer cells was measured. The results showed that the Poisson"s ratio of LNCaP cells was between 0.44 and 0.46, with an average value of 0.45. The influence of the location of the same cell feature points on the calculation results of Poisson"s ratio was within the error range of 1.6%. Conclusion This method is simple and feasible, can improve the measurement accuracy of Poisson"s ratio of cells, and is helpful for clinical cell detection and screening using cell mechanical properties.