The Countryside Alliance had its latest legal bid thwarted when three Appeal Court judges rejected its argument that the Hunting Act was unlawful.
The campaign group said it would now take its case to the House of Lords and to the European Court of Human Rights.
The RSPCA said the court decision shows the group's arguments are "wafer thin".
'Probably unprecedented'
The Countryside Alliance had claimed that the 1949 Parliament Act, which MPs used to introduce the Hunting Act after House of Lords opposition, is invalid.
What is the Parliament Act?
Hunt ban support 'declining'
It was appealing against a High Court ruling on 28 January that the act was clearly valid.
On Wednesday three Appeal Court judges described the challenge as "unusual, and in modern times probably unprecedented".
The alliance said it will make an application, probably next week, directly to the House of Lords to hear their appeal.
The law will now come into force on Friday although it is unclear whether prosecutions and arrests will follow.
The attorney general has ruled out a "blanket policy of non-enforcement", which the Countryside Alliance wanted to be put in place until all its legal avenues were exhausted.
In a statement, his office said: "The attorney general will, however consider with the director of public prosecutions and police what approach to take in relation to such prosecutions, and will be holding a meeting in the near future for that purpose."
After losing the appeal pro-hunt groups said the ban was unenforceable.
Simon Hart, of the alliance, said hunting would "look, sound and smell exactly the same" on Saturday because the police would not be able to enforce the law.
The alliance has said about 50,000 people are prepared to break the ban and continue hunting "in the full knowledge they will be arrested".
But the League Against Cruel Sports says it is setting up a "crimewatch service" to monitor the ban.
Its chief executive, Douglas Batchelor, said: "If we find criminal conspiracies to break the law then we will tell the police."
And RSCPA spokeswoman Becky Hawkes said it would also assist the police in bringing prosecutions.
