The relationship between the ellipticity of point spread function and the focal length of cylindrical lens was investigated. Fluorescent inverted microscopey imaging system is based on Olympus IX-83. With three different focal length,the point spread function with custom built stochastic optical reconstruction microscopy instruments was measured. A method to evaluate the cylindrical lens was developed based on linear region and localization error. The results show that linear region of 1μm and axial localization error of 16 nm can be achieved with correct focal length. As the demonstration,three dimension super-resolution image of Actin filaments was reconstructed.