Water, as one of the most important and indispensable small molecules in vivo, plays a crucial role in driving biological self-assembly processes. Real-space detection and identification of water-induced organic structures and further capture of dynamic dehydration processes are important yet challenging, which would help to reveal the cooperation and competition mechanisms among water-involved noncovalent interactions. Herein, introduction of water molecules onto the self-assembled thymine (T) structures under ultrahigh vacuum (UHV) conditions results in the hydration of hydrogen-bonded T dimers forming a well-ordered water-involved T structure. Reversibly, a local dehydration process is achieved by in situ scanning tunneling microscopy (STM) manipulation on single water molecules, where the adjacent T dimers connected with water molecules undergo a local chiral inversion process with the hydrogen-bonding configuration preserved. Such a strategy enables real-space identification and detection of the interactions between water and organic molecules, which may also shed light on the understanding of biologically relevant self-assembly processes driven by water.