Is it better to do it all at once? I'm very new to wxPython and I'm thinking it would be better to write the program in a way familiar to me, then apply the wxPython gui to it after I'm satisfied with the overall design of the app. Any advice?
IMHO, that would rather be a better idea. To keep the underlying business logic not tied down to the UI is a better approach that we can worry more about the underlying logic than bogging down too much about the interface. 
At the same time, it is also important to have some basic design for your interface so that it helps you have an idea about what kind of inputs and outputs are involved, and making the underlying logic support a wide range of inputs/outputs or simply wide range of interfaces.
Is it a bad idea to design and develop a python applications backend and then once finished try to apply a GUI to it?