Chelsea midfielder Barkley had been a doubt with an ankle injury while Aston Villa keeper Heaton is battling a calf problem.
Henderson, on loan at Sheffield United from Manchester United, has come in for Heaton but there are no plans to replace Barkley.
Heaton missed Sunday's 2-1 defeat at Wolves after injuring his calf in training while Barkley has sat out Chelsea's last six games.
An FA statement read: "Ross Barkley and Tom Heaton have been ruled out of England's forthcoming UEFA EURO 2020 qualifiers through injury.
"Goalkeeper Dean Henderson has been added to Gareth Southgate's squad in place of Aston Villa's Heaton but no replacements are planned for Chelsea's Barkley.
"Henderson will arrive at St George's Park on Monday as England's now 26-man squad begin preparations for their final fixtures of 2019."
Henderson has helped the promoted Blades to fifth in the Premier League after Saturday's 1-1 draw at Tottenham.
The 22-year-old also replaced Heaton in last month's squad after the Villa man pulled out.
England host Montenegro at Wembley on Thursday, where a win would book their spot at next summer's tournament.
Southgate's men then go to Kosovo for their final Group A match on Sunday.
England squad:
Goalkeepers: Dean Henderson (Sheffield United), Jordan Pickford (Everton), Nick Pope (Burnley)
Defenders: Trent Alexander-Arnold (Liverpool), Ben Chilwell (Leicester), Joe Gomez (Liverpool), Harry Maguire (Manchester United), Tyrone Mings (Aston Villa), Danny Rose (Tottenham), John Stones (Manchester City), Fikayo Tomori (Chelsea), Kieran Trippier (Atletico Madrid)
Midfielders: Fabian Delph (Everton), Jordan Henderson (Liverpool), James Maddison (Leicester City), Mason Mount (Chelsea), Alex Oxlade-Chamberlain (Liverpool), Declan Rice (West Ham), Harry Winks (Tottenham)
Forwards: Tammy Abraham (Chelsea), Callum Hudson-Odoi (Chelsea), Harry Kane (Tottenham), Marcus Rashford (Manchester United), Jadon Sancho (Borussia Dortmund), Raheem Sterling (Manchester City), Callum Wilson (Bournemouth)
Follow Sporting Life on social media - find us on Facebook here or tweet @SportingLifeFC
Related football links
Fast Results
Scores
