create your handytool.py: 

import sys
import pygtk
if not sys.platform == 'win32':
    pygtk.require('2.0')
import gtk

from mainwindow import MainWindow

if __name__ == '__main__':
    # enable threading
    gtk.threads_init()
    gtk.threads_enter()

    # create the main window
    myapp = MainWindow()

    # start the program loop
    gtk.main()

    # cleanup
    gtk.threads_leave()

create your setup.py file: 

from distutils.core import setup
import py2exe

setup(
    name = 'handytool',
    description = 'Some handy tool',
    version = '1.0',

    windows = [
                  {
                      'script': 'handytool.py',
                      'icon_resources': [(1, "handytool.ico")],
                  }
              ],

    options = {
                  'py2exe': {
                      'packages':'encodings',
                      'includes': 'cairo, pango, pangocairo, atk, gobject',
                  }
              },

    data_files=[
                   'handytool.glade',
                   'readme.txt'
               ]
)

run setup.py py2exe. You'll get a warning: 

The following modules appear to be missing
['gdk', 'ltihooks']

Ignore it ;)

Once that's done, you'll need to copy the etc, lib and share directories from your GTK+ install to the dist dir py2exe created. Optionaly, you can clean the share\locale dir to include only the locales you need for GTK+. Same thing for share\themes (I left both Default and MS-Windows).

That's it :D

Tested with: