He is a large, circular humanoid with "punch-holes" for eyes, light-brown skin covered with multiple warts, and stubby limbs. He sports with a autopsy stitch from his chest. His mouth with crooked teeth-filled jaw is larger enough to be filled with swarm of flies.
From the original game, the Duke's is colored light-brown. However in Rebirth, his color is now periwinkle.
He was changed back in Repentance.
The Duke coughs out regular Flies and Attack Flies around him and floats around the screen in random directions, while bouncing off of walls. He takes a large amount of knockback from Isaac's tears.
The Duke coughs out regular flies for defense and will keep doing so if Isaac continue destroying them. These flies will circle around him. If he has too many flies around him, he can blow them away at high speeds, which will cause them to follow Isaac.
Once he has sufficient defensive flies, he will proceed to spawn attack flies instead, which are stronger and poised to attack the player from the start.
Upon death, he spawns a single Pooter and 2 more Attack Flies. In Rebirth, however, no longer does so.
Actually getting Isaac's shots through to The Duke of Flies is the most challenging part of this encounter, and that can be accomplished in a few main ways:
Firing through the gaps in his shield will make it easier to kill him.
A single Bomb will clear out most of the flies, and deal a very large amount of damage to him.
The boss gets significantly harder if Isaac can't land his shots right away. The green champion variation is particularly dangerous as shooting down Moters will make it very easy to get hit.
When the Duke starts spitting up large Attack Flies, focus on them when they appear to keep things under control.
If Isaac get swarmed by the Flies, it is always a good idea to leave the Duke alone and concentrate on his spawns. While this will not damage the Duke in any way, ignoring the flies for too long can lead to so many of them that they become too much to handle.
Having Skatole will grant immunity against all flies that are not orbiting the Duke. Attack flies that have been spawned by the Duke but not yet released from his orbit will harm Isaac if he runs into them.
Having Bloody Lust will allow the player to stack up a large amount of damage by killing flies, thus being able to one-shot them in a short time and deal high damage to the Duke. Because of this, Samson is probably the best character to use against this boss.
The Duke of Flies has three distinct sounds that he uses before an attack. Learning what these are will help when fighting him.
One sound for spawning a large Attack Fly (when he smiles).
One sound for spawning 3 Attack Flies (when he swell his cheeks).
One sound for releasing some defensive flies to attack Isaac.
Larger and moves slower than the original Duke of Flies. Instead of spawning large Attack Flies, he spawns Suckers.
Shoots blood in all directions when he coughs, and spawns Moters instead of Attack Flies.
From one of The Duke of Flies' sketches in the artbook, his back view (which shows his visible butt-cheeks) can be seen. It was never displayed in the game, however.
The teeth from his upper jaw are going to be visible.
His eyes are originally X-shaped rather than looking like punch-holes.
The name "the Duke of Flies" is not a reference to Beelzebub, Edmund was unaware of this connection and simply chose the name because it "sounded neat".
The protective ring of insects the Duke spits out to surround himself with is referred to as the Halo Of Flies, just like the item Isaac can pick up to protect himself with orbiting flies.
The Duke was confirmed to be Edmund McMillen's portrayal of the Pokémon Koffing.
He appear in the original game's first trailer.
The stitching below the Duke's mouth appears very similar to an autopsy stitch - a Y-shaped incision that allows access to the cadaver's organs that is then stitched back up when the autopsy is complete.
In Rebirth, flies from inside The Duke of Flies' mouth aren't animated at all for some reasons, unlike in the original game.