Ashton met Morsi "for two hours," her spokeswoman Maja Kocijancic told AFP.
In Cairo to discuss the bloody political crisis gripping the Arab world's most populous state, Ashton headed off to meet Morsi during the night.
Sources told AFP that she had left Cairo in a military helicopter for the undisclosed location where Morsi is being held.
On her last visit on July 17, Ashton unsuccessfully requested to meet him and urged his release.
Ashton, who arrived in Cairo late Sunday, held talks with a string of officials throughout yesterday, including military-installed interim president Adly Mansour, vice president for international affairs Mohamed ElBaradei and army chief General Abdel-Fattah al-Sisi.