Madhuku was born on 20 July 1966 in Chipinge, Zimbabwe. He attended the University of Zimbabwe (UZ), receiving a Bachelor of Law degree in 1990. He then travelled to the UK to study at the University of Cambridge, receiving a Master of Law in 1994 and a doctorate in 1999. In 2010, he published a book titled An Introduction to Zimbabwean Law. He was made a full professor at UZ in 2011.
Madhuku is a founding member of the National Constitutional Assembly (NCA), a pro-democracy group allied with the Movement for Democratic Change led by Morgan Tsvangirai. The group opposes the one-party rule of President Robert Mugabe and seeks to establish a democratic constitution. Madhuku served as its vice President from 1997 to 2001 and its President from 2001 to 2011. In 2000, he helped to defeat a constitution introduced by Mugabe in a national referendum. Mugabe described Madhuku's activities as "opportunism", stating: "There are some fraudulent human rights campaigners like Lovemore Madhuku and his NCA who, when broke, intentionally provoke the police in order to get arrested and raise money from the donors. As such, they easily attract the attention international media line CNN, BBC over nothing. That's the Madhuku survival strategy for you".
In November 2001, Madhuku was detained without charge for leading a demonstration after Soldiers allegedly strangled a student and threw him from a train. According to Journalist Geoffrey Nyarota, Madhuku was also subject to a smear campaign by state-owned media. Madhuku has stated that his country home was burned down and his house in Harare badly damaged by attacks. In February 2004, he was arrested during a protest, beaten, and left for dead outside Harare. On recovering, he stated, "We will not be deterred by the beatings and the cruelty of this regime. They can only stop us by killing us." In October of the same year, Mugabe's government introduced a bill into parliament seeking to ban nongovernmental organizations, including the NCA. In November 2006, he was charged with organizing an illegal protest, but a magistrate later dismissed the charges. Police assaulted him again in March 2007, breaking his arm and leaving him with cuts to the head and body.
Madhuku was re-elected as the NCA's chair in 2006 under controversial circumstances, as he had amended its constitution to extend his term of office. According to Radio Netherlands, Madhuku was particularly criticized for serving several terms after having himself criticized Mugabe for serving more than two terms in office. He completed his final term as the group's chair in 2011.
He was summoned to court again in 2011 for his leadership of 2004 protests, drawing international criticism.