In this study, the degree of alignment of polymer microfibers produced by electrospinning using a rotating water collector was evaluated. Aligned micro- and nano-fibers are required in various practical applications involving anisotropic properties. The degree of fiber alignment has many significant effects; hence, and accurate quantitative analysis of fiber alignment is necessary. Therefore, this study developed a simple and efficient method based on two-dimensional fast Fourier transform, followed by ellipse fitting. As a demonstrative example, the polymer microfibers were electrospun on the rotating water collector as the alignment of microfibers can be easily controlled. The analysis shows that the flow velocity of the liquid collector significantly affects the electrospun microfiber alignment, that is, the higher the flow velocity of the liquid collector, the greater is the degree of microfiber alignment. This method can be used for analyzing the fiber alignment in various fields such as smart sensors, fibers, composites, and textile engineering.