The troubleshooting you've done on your own so far to resolve saving your cheque alignment for printing is great. Cheque alignment can be tricky, and it sounds like you're already familiar with the process, but now it's not saving. It's important to me you're able to save your settings and I'm here to help.
It can take a few tries and a bit of finessing, but once you hit OK or Finish Setup, depending on whether you're trying to align payroll or expense cheques, QuickBooks should remember that setup for next time. When you say it won't save, I take that as either meaning those two buttons I just mentioned aren't responsive or that despite saving, next time you print a cheque, you have to go through the process again. I'd like to start you off with some troubleshooting steps.
The first troubleshooting we do as technical support for this program is browser or app troubleshooting. As a cloud-based software, the data collected from the Internet plays a big role in QuickBooks Online's overall functionality. Temporary Internet files can cause strange behaviours and prevent things from working as they should, so here are the Internet related steps I always recommend.
If after all that you're still having trouble saving your preferences, get in touch with a QuickBooks Online tech support agent using one of the following methods.
Phone and chat: ClickContact Us to learn how to reach phone and chat support.
Schedule a Callback: click (?)Help in the upper right > click Contact Us > explain your situation > click Let's Talk > choose Get a callback (Note: Callback hours are the same as regular phone hours.)
Social Media: Facebook and Twitter from Monday to Friday between 9 a.m. and 8 p.m. ET
For your reference, here are a couple articles about cheque printing and alignment.