Perhaps "emphasis" isn't the most helpful word for OP to use here. The example statement...

1: He does know the truth _{(there's absolutely no doubt about that!)}

...would be very unusual if it was simply intended to convey the "emphasis" of, for example,...

2: He really knows the truth!

Normally, the "do-support" of #1 above to refute / contradict an earlier assertion, such as...

3a: "I doubt if even John knows the truth of the matter."
3b: "You're mistaken. He does know the truth."

Note that the word does will always carry heavy stress in such contexts.

By the same token, in OP's question context, including do-support, as...

4: Who does know the truth?

...would normally only occur in contexts where the speaker has just been told that someone he thought might know the truth actually doesn't - equivalent to...

5: If what I previously thought isn't true, then what is true?

OR

6a: "It's no use asking me. I don't have a clue!"
6b: "Who does know the truth, then?"

Perhaps "emphasis" isn't the most helpful word for OP to use here. The example statement...

1: He does know the truth

...would be unlikely (but not impossible) if it was simply intended to convey the "emphasis" of, for example,...

2: He really knows the truth! _{(there's absolutely no doubt about that!)}

Normally, the "do-support" of #1 above refutes / contradicts some earlier assertion, such as...

3a: "I doubt if even John knows the truth of the matter."
3b: "You're mistaken. He does know the truth."

Note that the word does will always carry heavy stress in such contexts.

By the same token, in OP's question context, including do-support, as...

4: Who does know the truth?

...would normally only occur in contexts where the speaker has just been told that someone he thought might know the truth actually doesn't - equivalent to...

5: If what I previously thought isn't true, then what is true?

OR

6a: "It's no use asking me. I don't have a clue!"
6b: "Who does know the truth, then?"