Can you access/farm the secret level in Diablo 3 more than once per difficulty?
According to the wiki at IGN, you can run the secret area as many times as you like. This has been confirmed several times since, including this video. Once you have a Staff of Herding for a difficulty, you can enter the secret area as many times as you want.
You can use the staff from a higher difficulty to open the secret level in a lower difficulty so you can feel free to create the Hellish or Infernal Staff of Herding when you're able.
I have accessed Whimsyshire twice with two different characters. It seems all you have to do is put the Staff back in your chest and equip it, find the cow's ghosts and you're good to go. All you need is to upgrade depending the difficulty level. For instance, I have the Nightmare one. Not disappearing anymore.