An image analysis processing method for the measurement of nanofiber diameter was developed. For the analysis, scanning electron microscopy (SEM) images of electrospun fiber were prepared and applied to the individual measurement of the fiber diameter by using the developed and the traditional manual methods. Both methods provided a similar fiber distribution. The fiber average diameters were similar but the variance of the new method was larger than that of the manual method. The average diameters from the two methods exhibited a linear relationship with a high coefficient. The developed method can be used as a practical tool to estimate the fiber diameter of the electro spun web.