RNA-coat protein interactions in intact tobacco mosaic virus have been investigated for the first time directly on the single-molecule level by pulling the genetic RNA step by step out of the helical groove formed by its protein coat. The effects of pulling speed and pH on RNA-protein interactions are presented. In addition, the rebinding behavior of the detached RNA with the protein coat is discussed. Our results demonstrate the possibility of studying nucleic acid-protein interactions in more complicated systems using AFM-based single-molecule force spectroscopy.