The order from Chief Minister J. Jayalalithaa came a day after the Supreme Court commuted the death sentence handed down to three of the seven people convicted over their role in the 1991 assassination in her state.
But in his first intervention in the case, Singh said the release of the seven "would be contrary to all principles of justice".
"The assassination of Shri Rajiv Gandhi was an attack on the soul of India," Singh said in a statement.
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"No government or party should be soft in our fight against terrorism," he added.
Singh confirmed in his statement that the government had lodged a petition before the Supreme Court to challenge the release which is expected to be heard by judges later Thursday.
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Rajiv Gandhi, whose widow Sonia is now president of Singh's Congress pa