AIADMK coordinator O Panneerselvam and co-coordinator E Palaniswami announced "removing" 44 functionaries from the party's primary membership.
Those who faced the action included R Samy, a former MLA from Melur in Madurai and a staunch supporter of Mr Dhinakaran.
In a joint statement, Mr Panneerselvam and Mr Palaniswami named the functionaries, holding various posts including membership to the powerful decision making General Council and covering the AIADMK units in Madurai, Villupuram, Dharmapuri, Tiruchirappalli and Perambalur among others.
They were being "removed" since they went against the party's principles and brought "disrepute" to the AIADMK, the two leaders said in the statement.
"Party workers are requested not to have any truck with them," Mr Palaniswami, also the Chief Minister, and his deputy Mr Panneerselvam, said in the statement.
Further, Mr Panneerselvam and Mr Palaniswami "relieved" two others from party posts in Puthukottai and Vellore districts.
Stung by the RK Nagar by-election defeat, the ruling AIADMK on December 25 cracked the whip against nine party functionaries. The two leaders had then sacked four of Mr Dhinakaran's aides as district secretaries, while expelling five others.
In a jolt to the ruling camp, TTV Dhinakaran had won the December 21 RK Nagar by-election against its candidate E Madhusudhanan by a thumping margin of 40,000 votes.
The by-election, necessitated due to the death of late chief minister J Jayalalithaa who was the sitting MLA, was seen as a litmus test for the 20-month-old AIADMK government.
Mr Dhinakaran, sidelined by the ruling camp a few months ago, had won as an independent candidate.
He had earlier lost his battle for the party symbol 'Two Leaves' to the Palaniswami-led ruling camp and contested the election on the 'Pressure Cooker' symbol.