“I know that some of my many colleagues aren’t happy that I am breaking with Senate tradition to testify on the nomination of one of my colleagues, but I believe, like perhaps all of my colleagues in the Senate, that in the choice of standing with Senate norms or standing up for what my conscience tells me is best for our country, I will always choose my conscious and country," Booker said.
Booker said he believed President-elect Trump's pick for attorney general has "not demonstrated a commitment" to a central prerequisite of the job -- to demand equal rights and justice for all citizens.
"In fact, at numerous times in his career, he has demonstrated a hostility towards these convictions," Booker said of Sessions.
The New Jersey senator said he was "deeply motivated" by the many issues the next attorney general will influence, especially the crisis of mass incarceration.
Booker said that while he and Sessions disagreed on the issues, they have always exercised a "collegiality and a mutual respect." In fact, Booker spoke of legislation that he and Sessions co-sponsored to award the Congressional Medal of Honor to the "foot soldiers who marched at Selma."
It was an emotional plea in which Booker said that those marchers in Selma inspired him as a young lawyer to seek justice for all in New Jersey, and begin representing black families looking to integrate white neighborhoods who were turned away and denied housing.
"I am literally sitting here because of people, marchers in Alabama," he said.
Booker said that his colleague's record "indicates that he won't" pursue justice for women, defend the equal rights of gay and lesbian and transgender Americans, defend voting rights or defend the rights of immigrants as attorney general.
This was the first time a sitting senator testified against another sitting senator in a cabinet confirmation hearing, according to Booker's office, which contacted the Senate Historian's office for the records.
Civil rights icon Rep. John Lewis, D-Georgia, who helped lead the 1965 march in Selma, and Cedric Richmond, D-Louisiana, leader of the Congressional Black Caucus, also expressed concern about Sessions' nomination at today's hearing.
"It doesn’t matter how Senator Sessions may smile, how friendly he may be, how he may speak to you, but we need someone who is going to stand up, speak up and speak out. For the people that need help for people who have been discriminated against," Lewis said.
He told the senators that after decades of progress, many Americans are concerned that some leaders want to return to the "dark past, and the power of the law will be used to deny freedoms protected by the Constitution."
"We need someone as attorney general who is going to look out for all of us and not just for some of us," Lewis said.
However, others on today's panel came to the Alabama senator's defense.
"A lot has been said about Senator Sessions' character. We have seen people who have never met Senator Sessions claim to know him and know his heart. We have seen members of this body and members of the House of Representatives just now who have worked with Senator Sessions and praise him for his work and now attack him. This should not be," said Sessions' former staffer William Smith.
Smith, the former Chief Counsel on the Administrative Oversight and the Courts Subcommittee, said that his former boss had fought for civil rights and against the Ku Klux Klan and fought for people regardless of the color of their skin.
"I'm not testifying to someone who just met him yesterday. I've known his family, I've dined at his house. We've eaten Johnny Rockets burgers together," Smith added. "I've been in every political situation with him. Senator Sessions is unquestionably qualified for the job for which he has been nominated. He is a good Christian man, and a good family man. He is a man who has dedicated his public life to service."
ABC News' Hayley Walker and Ben Siegel contributed to this report.