In order to unshare real serial port in your system, please, follow these steps:
1. Important notice: close all shared serial port which you want to unshare prior to unsharing.
2. Launch "Shared Serial Ports" application in case it is not started yet. For instance, the default path to it is: "Start" (Windows button) -> "All programs" -> "Eltima Software" -> "Shared Serial Ports" -> "Configure Shared Serial Ports".
3. Select serial port you want to be unshared in Serial ports tree.

4. Click "Unshare port" Toolbar button.

Note: you cannot unshare serial port which is not shared.
See Also:
How to share real serial port?
How to unshare all serial ports?