Dressed in a dark suit and a maroon tie, Assange confirmed his identity at the beginning of the hearing and formally declared that he rejected extradition. It was his first public appearance since February, when a first week of hearings were held before the case was postponed.
The Australian, 49, was due to return to court in April but the process was delayed by the coronavirus pandemic.
The hearings are now expected to last three to four weeks and be marked by protests.
The United States charges him with espionage for releasing more than 700,000 classified documents on US military and diplomatic activities in 2010, mostly in Iraq and Afghanistan, which revealed acts of torture, civilian deaths and other abuses.
In recent weeks, the United States has presented new evidence against the WikiLeaks founder, accusing him in particular of having hired hackers.
Held in a high-security London prison since his arrest in April 2019 at the Ecuadorian embassy, where he lived for seven years, Assange could face 175 years in prison if found guilty by US justice.
But for his supporting committee, they are "politically motivated charges" that "represent an unprecedented attack on press freedom."
Before ruling, the English courts must ensure that the United States' request is not disproportionate or incompatible with human rights.
His lawyers have tried to have the new accusations dropped from the proceedings, arguing that he had not had time to prepare, since communication with his client has been difficult due to restrictions related to the pandemic.
What happens is abnormal and liable to create an injustice, denounced his lawyer Mark Summers.
However, they managed to convince Judge Vanessa Baraitser to be analyzed "during the examination of the extradition request and not before."
Several dozen people, including British designer Vivienne Westwood, demonstrated outside the courthouse on Monday with banners reading "Imprison War Criminals, Free Julian Assange!"
"The future of journalism is at stake," said WikiLeaks editor-in-chief Kristinn Hrafnsson, while Assange's father, John Shipton, denounced an "abusive trial."
Assange's partner, Stella Moris, who says she fears that Assange will take his own life, delivered a petition with 800,000 signatures against his extradition to the British prime minister's office.
Allowing the extradition of Julian Assange on this basis would have a chilling effect on freedom of the press and ultimately could hamper the work of the press as a provider of information and public guardian in democratic societies, warned the Council of Europe.
United States defends that Assange is not a journalist but a "hacker" and claims that he helped intelligence analyst Chelsea Manning steal these secret documents before exposing the material to the eyes of the world.
US also accuse him of having conspired with members of the hacker groups LulzSec and Anonymous and of having had unauthorized access to a government computer system of a NATO country.
Assange's lawyers have been warning for months about the fragile physical and mental state of the Australian, who in previous appearances appeared confused and with difficulties to express himself or was simply absent due to health problems.
In the February hearings they assured that US President Donald Trump had promised to forgive him, if he denied that some emails published by WikiLeaks and that contributed to the defeat of his Democratic rival Hillary Clinton in the 2016 presidential elections had been leaked by Russia.
The White House has denied this accusation.
According to the Australian's defense, Trump wants to make an example case of Assange in his "war against investigative journalists" and Assange would not have a fair trial in the United States.
"The political persecution is evident," said former Spanish judge Baltasar Garzón, who coordinates his team of lawyers internationally, on the sidelines of the February hearings.