The left-hander had been a doubtful starter for his home-town Test after injuring his hand during his spectacular debut in the series-clinching six-wicket win in Mohali last week. Dhawan blazed 187 from 174 balls - the highest ever score by an Indian debutant - while his 85-ball ball century was the fastest in Test history.
The 27-year-old did not reappear for India's chase, however, after he injured his finger attempting to field a Phil Hughes cut shot during Australia's second innings on the fourth day. Suresh Raina has been called in as cover after replacement opener Gautam Gambhir was diagnosed with jaundice.
"Injured Shikhar Dhawan ruled out of Delhi Test," the Board of Control for Cricket in India announced on their Twitter page. "@ImRaina included in the squad; @GautamGambhir misses out due to jaundice #IndvAus."
Dhawan's injury means that India will be forced into another reshuffle at the top of the order after Dhawan had been brought in to replace out-of-form veteran Virender Sehwag.
Gambhir had loomed as an obvious like-for-like replacement, however, his illness means India are left with Murali Vijay and Ajinkya Rahane as their recognised opening batsman as they attempt to claim a 4-0 series sweep.
The fourth Test begins at the Feroz Shah Kotla Stadium in Delhi on Friday. A statement from the BBCI revealed that Dhawan had suffered a fracture in his left hand that will also sideline him for the start of the upcoming Indian Premier League, which begins on April 3.
"Shikhar Dhawan was ruled out of the fourth AIRTEL Test against Australia - after sustaining a fracture in his left hand. He will be out of action for six weeks," the statement said.
"The All-India senior selection committee then added Gautam Gambhir and Suresh Raina to the squad for the fourth Test.
"However, Gambhir has been diagnosed with jaundice, after undergoing a blood test, earlier today (Tuesday). The official announcement of the team was delayed, as the board was waiting for the copy of the report. Gambhir will not take part in the Delhi Test, as a result."