Objective To propose a method for determing cell Poisson’s ratio based on micropipette aspiration technique. Methods 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 position changes of markers on the surface of cell membrane. ResultsThe Poisson’s ratio of LNCaP cells in prostate cancer cells was measured. The result 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 calculation results of Poisson’s ratio was within the error range of 1.6%. Conclusions This method is simple and feasible, can improve the measurement accuracy of Poisson’s ratio of cells, and is helpful for cell detection and screening by using cell mechanical properties in clinic.