The Tykes fielded an ineligible player for the first round replay with Horsham, a match that they went on to win 3-0.
The League One side were due to face a trip to Sutton United on December 2 in the second round of the competition, however it'll now be the Isthmian League Premier Division side who take their place in the next round.
A statement from the FA said: "A Professional Game Board Sub-Committee has removed Barnsley from the 2023-24 Emirates FA Cup for fielding an ineligible player during their First Round Proper Replay against Horsham on Tuesday 14 November 2023.
The FA alleged that the player was ineligible for this replay, as he was not correctly registered and eligible for the original First Round Proper match on Friday 3 November 2023, which constitutes a breach of FA Cup Rule 103.
"Barnsley admitted this charge and acknowledged that the breach had occurred.
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"The Sub-Committee's members ordered that Barnsley be removed from the competition, and that Horsham be awarded the tie and progression to the Second Round Proper.
"Subject to any appeal by Barnsley, Horsham will now play away to Sutton United in the Second Round Proper on Saturday 2 December 2023."
More to follow...