Auto focusing is key technique in ellipsometric imaging system. The process is studied by use of several autofocus' evaluation functions. FIB on Si wafer and multi-channel proteinchip has been used in experiment. The change of functions value has been studied. The effect of noise and selected area used for auto focusing are evaluated. The experiment shows that Laplacian function and Sobel function are suitable for this system. By using several frames to get average result, the effect of noise has reduced to good level when the number of frame more than 9. Along the same sharp edge, selecting different areas for autofocus, it has no influence on result. Using autofocus evaluation function to get the focus image of tilting plane, and the result agrees with the theoretical plane of image. It has been confirmed that autofocus evaluation function can be used in ellipsometric imaging system.