The group played 16 songs including classics such as 2 Minutes to Midnight, Fear of the Dark and The Number of the Beast.
It was also the final night of their Maiden England world tour at Knebworth.
"Three years of touring comes to an end tonight," said lead singer Bruce Dickinson.
"This is the culmination of those three years. We should be pretty good by now, if we don't get it right tonight then we never will."
Dickinson finished by hinting at a new album: "What's coming in the next few years is going to make it more real for you guys, believe me."
Long-time mascot Eddie made several appearances through the band's set including a giant version that walked across the stage during Run to the Hills.
Earlier in the evening, Bruce Dickinson took part in a mock dog fight above the grounds of Knebworth House to commemorate the start of World War One.
Dickinson flew one of the planes in the display, which lasted for 15 minutes and ended with the Band and Bugles of The Rifles playing the Last Post just before Deftone's set on the main stage.
Slayer paid tribute to former guitarist Jeff Hanneman during their final track Angel of Death on the second stage.
Hanneman died of liver failure last May.
There was also a big crowd for former Skid Row singer Sebastian Bach in the main tent.
All the entrances were shut about 20 minutes before the performance with the area at full capacity.
One group of angry festival-goers tried to push a security barrier over during the set after being denied access.
Festival favourite Frank Turner played on the main stage and said his friends had asked him if he was nervous about playing a heavy music festival.
"I said 'Of course I'm not nervous because this is where I come from; rock, punk and heavy metal.'
"By the way, for those of you who don't know what this is, it's an acoustic guitar," he told the crowd.
"It might be an acoustic guitar but I'm going to die with a BC Rich Warlock in my hands."
Amber Paul, 18, Nino, 17, Megan, 16, Summer, 16, Hannah, 17, and Ashley Wiggans, 18, all met on Twitter a few years ago.
They're camping for the weekend and said they enjoyed the way the stages were set out.
"It's a small site, so there's less walking," said Hannah. "And there are no clashes."
There are two main stages at Sonisphere and as a band finishes on one, another act starts on the other.
One of the more surreal moments of the festival was provided by Japanese metal outfit Babymetal, who were playing their first gig in the UK.
The group is made up of three teenage, female singers and a backing band who were dressed in white robes and facepaint.
In an introduction video called Metal Resistance: Episode II, a parody of the opening credits of Star Wars, the band revealed they were the self-styled saviours of heavy metal.
They also paid tribute to "metal gods" Iron Maiden and later in their set, Metallica guitarist Kirk Hammett.
There were also performances from Blitz Kids, Vodoo Six, The Sisters of Mercy, Anthrax, Ghost and The Virginmarys.
Sunday is headlined by Metallica, whose set By Request has been chosen by fans, with performances elsewhere from Alice in Chains, Mastodon, Airbourne and Krokodil, the band featuring Radio 1 rock show host Daniel P Carter.
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Metal band Iron Maiden pulled in the biggest crowd of the weekend so far as they headlined the Saturday night at this year's Sonisphere Festival.