>>1
Which 16bit exes? DOS EXE or NE EXE?
32bit exes are PE, and quite different from DOS exes, altough they are backward compatible (a PE is usually also a DOS exe, but the DOS code is never ran when you're using it in a 32bit OS. Running such Windows PE files in DOS will result treating it as a DOS EXE, which code usually just displays a message like "This program cannot be run in DOS mode."
You'll have to study the PE file format if you want to know how to make your own, or alternatively, you could just use Microsoft's Assembler and Linker, and just generate the assembly listing, which is likely less work, and it's usually how most people proceed. If you want portability, you could make it compatible with some open source assemblers/linkers and target more OSes, altough you'll have to provide OS-specific runtime as well.