Yes, if you are using Turbotax Online, you need a second account with a separate user name and password, and you will be charged a separate fee, unless you qualify for free filing. Each online account can only prepare one return.
When married filing separately, it is often more cost effective to buy Turbotax as a download to install on your own computer, since the desktop version can e-file up to 5 returns. (Additional fees sometimes apply, but unless you qualify for free online filing, the desktop version is still usually the same or less than filing two online returns.)