The Chelsea star, who captained his club to Champions League glory for the first time the weekend before last, pulled his thigh while chasing a loose ball at London Colney on Wednesday.
He was ruled out of the tournament in Poland and Ukraine on Thursday after a scan and subsequent examination by the England medical staff.
Liverpool’s Jordan Henderson replaces him in the squad, subject to agreement from UEFA’s medical team.
“It’s a huge blow for the team, especially coming after the loss of another senior player in Gareth Barry,” said England manager Roy Hodgson.
“The fact that the doctor cannot guarantee Frank could take part in any of the group matches means we have to replace him in the central midfield area.
“Apart from it being a huge blow to me and the team, I’m especially disappointed for Frank himself. He has been in very good form and was looking forward to the tournament.
"He made a huge contribution towards us qualifying for the Euros so it is cruel to be forced out through injury just before the tournament.”
Versatile Everton defender Phil Jagielka replaced Barry in the 23-man squad after the Manchester City midfielder tore a muscle in his lower abdomen during Saturday's 1-0 friendly win over Norway in Oslo.
England's hopes were also earlier hit by the long-term absence of gifted Arsenal playmaker Jack Wilshere.
Lampard had returned to the fore with Chelsea following the replacement of Andre Villas-Boas with Roberto Di Matteo, and helped the Blues win the FA Cup before leading them to an unlikely European Cup triumph over Bayern Munich in the Bundesliga side’s Allianz Arena home.
Club captain John Terry was suspended for the final after his red card for kneeing Alexis Sanchez in the semi-final against Barcelona.
Speaking prior to his injury, Lampard, who turns 34 next month, said: "I did wonder if I was going to make it (the Euros) earlier in the season. I'm not silly. I wondered if I'd be out.
"I took nothing for granted at that stage and it just shows you how football can turn, not just for myself.
"A few in the squad might have been out of the picture a few months ago and now they are in the picture. I can only be thankful that I just kept going through those times."
England’s preparations for the finals have been lower-key than for any tournament in recent times, with only a few thousand fans expected to make the trip to Eastern Europe, confidence in the team at a low level and Hodgson – regarded as a failure at Liverpool earlier this season – appointed in place of Harry Redknapp following the departure of Fabio Capello.
England play Belgium in a friendly at Wembley on Saturday then open their Euro 2012 campaign against France at the Donbass Arena in Donetsk on June 11. They then play Sweden in Kiev on June 15 before the final group game against hosts Ukraine, again at the Donbass, on June 19.