On-loan Hearts striker Euan Henderson led the attack in the Spiders' 2-0 win in Tuesday's rescheduled tie at the Caledonian Stadium but the 22-year-old should not have played as he was not signed prior to the original date of the game (January 21) before it was postponed due to the weather.
Henderson joined Queen's on January 27 and Scottish Cup rules say that players can only play if they are registered before the original date of the game.
Inverness have been reinstated to the tournament and will now travel to Livingston in the fifth round next weekend.
A statement from the Scottish Football Association on Friday said: "Following a disciplinary tribunal, Queen's Park have been ejected from the Men's Scottish Cup for a breach of Competition Rule 12.5, relating to player eligibility.
"Livingston will now face Inverness Caledonian Thistle in the Fifth Round."
Queen's Park, the cinch Championship leaders, have acknowledged their mistake and accepted the decision.
"An error was made regarding the eligibility of a player who featured in Tuesday night's game with Inverness Caledonian Thistle and we have accepted the SFA's decision," said club president David Hunter.
"It is a regrettable mistake. One we will learn from. Our team put on a tremendous display on Tuesday to win the tie and obviously today's result is a hugely disappointing outcome.
"As upsetting as this matter is, it's vital that we put this behind us quickly, move on and focus on our league campaign.
"The support we received from our fans on Tuesday was terrific. We are conscious that they invested time and money travelling to the game.
"I will be speaking to the Supporters Association over the coming days to suggest how the club can recognise the efforts of the travelling support."