Amnesty head Salil Shetty made the appeal during a meeting with European Union diplomatic chief Federica Mogherini in Brussels, while protesters gathered outside with giant placards bearing names of the detained activists.
"We believe the arrest of both the (local) chairman and director of Amnesty International signifies a step change in the human rights crisis in Turkey," Shetty said after meeting Mogherini.
"The EU has to make it clear that they have definitely crossed a red line and that it requires a resetting of the relationship with the EU."
Shetty said he had asked for the meeting with Mogherini to press Amnesty's message that the EU should no longer tolerate Ankara's actions.
The coup "has become an excuse to lock up anybody asking a question, starting a debate," Shetty told reporters at the protest outside the EU headquarters.
"This is totally beyond the pale; for the sake of the Turkish people, for Europe, Turkey has to be brought back in line and this is the moment to do it," he said.
Turkey began formal membership talks in 2005 after years of foot-dragging by EU member states wary of admitting such a large Muslim country.
Progress was slow but the negotiations came to a virtual halt last year as Turkish President Recep Tayyip Erdogan rounded up opponents in a massive crackdown.
The situation is further complicated by an agreement reached in early 2016 to halt the flood of migrants and refugees pouring through Turkey to Greece and then onto other parts of Europe.
Germany took in nearly one million refugees, mostly fleeing the war in Syria, and there have been growing concerns that a complete breakdown in relations with Turkey could scupper the deal and cause a fresh migrant crisis.
Shetty said Turkey's role in helping the migrants was "always appreciated" but it also allowed Anakara "to hold the EU hostage."
German Chancellor Angela Merkel faces elections in September and the migrant issue is widely seen as one of the most delicate she faces.
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