Hello wuliao23,
The following discussion forum post describes a few methods to track movement in images : http://forums.ni.com/ni/board/message?board.id=200&message.id=14307&requireLogin=False.
You could also consider using the operators such as 'IMAQ Subtract', which can be found in the 'Image Processing' section of the 'Vision and Motion' functions palette, to detect motion of one object in the video, by comparing consecutive frames of a video file.
Could you elaborate on the second problem you were having? How exactly did you draw a line for ROI? Was it using the Vision Assistant? What were you trying to do with the pixel values?
Vivek Nath
National Instruments
Applications Engineer
Machine Vision