The detection of the precise movement of a vesicle during transport in a live cell provides key information for the intracellular delivery process. Here we report a novel numerical method for analyzing three-dimensional vesicle movement. Since the vesicle moves along a linear cytoskeleton during the active transport, our method first detects the orientation and position of the cytoskeleton as a linear section based on angle correlation and linear regression, after noise reduction. Then, the precise vesicle movement is calculated using vector analysis, in terms of rotation angle and translational displacement. Using this method, various vesicle trajectories obtained via high spatiotemporal resolution microscopy can be understood..