A groin injury means Reus joins Julian Brandt, Sebastian Rudy and Karim Bellarabi to make up the four names cut as Germany announced their final 23-man squad while injured captain Bastian Schweinsteiger, who tore his knee ligaments in March, is included.
Reus, Borussia Dortmund's fleet-footed winger, missed out on Germany's 2014 World Cup win after tearing ankle ligaments in a warm-up friendly international just before the squad flew to Brazil.
Now an injury on club duty has scuppered his bid to be fit for the finals in France.
Germany coach Joachim Löw has opted not to take Reus, although both Borussia Dortmund's Bayern Munich-bound centre-back Mats Hummels and Manchester United midfielder Schweinsteiger are included even though both are carrying injuries.
“The medical department have confirmed that Bastian Schweinsteiger and Mats Hummels will be fit to play at the tournament,” explained Löw at Germany's pre-Euro training camp in Ascona, Switzerland.
“The prognosis for him [Reus] wasn't good, so for medical reasons we had to strike him off the list.
“Of course, it's bitterly disappointing for both him and for us.”
Reus' former Dortmund team-mate Mario Götze, whose goal won the 2014 World Cup final against Argentina, tweeted: “Happy Birthday, Marco, but we are all really sad. Unbelievable. That hurts me.”.
Löw has included two promising 20-year-olds in Dortmund defensive midfielder Julian Weigl and Schalke forward Leroy Sane, who both made their debuts in Sunday's 3-1 friendly defeat to Slovakia.
Bayer Leverkusen's Brandt, 20, who also won his first cap against Slovakia, misses out, while his club colleague and fellow winger Bellarabi, who has won 10 caps, is ruled out with injury.
Hoffenheim midfielder Rudy completes the quartet to be left at home as Germany now turn their attentions to their final pre-tournament friendly on Saturday in Gelsenkirchen against Hungary.
The Germans open their Group C campaign against Ukraine in Lille on June 12 before also playing Poland and Northern Ireland.
Germany's 23-man squad for Euro 2016:
Goalkeepers: Manuel Neuer (Bayern Munich), Bernd Leno (Bayer Leverkusen), Marc-Andre ter Stegen (Barcelona/ESP)
Defenders: Jerome Boateng (Bayern Munich), Emre Can (Liverpool/ENG), Jonas Hector (Cologne), Benedikt Hoewedes (Schalke 04), Mats Hummels (Borussia Dortmund), Shkodran Mustafi (Valencia/ESP), Antonio Ruediger (AS Roma/ITA)
Midfielders: Julian Draxler (Wolfsburg), Sami Khedira (Juventus/ITA), Joshua Kimmich (Bayern Munich), Toni Kroos (Real Madrid/ESP), Mesut Özil (Arsenal/ENG), Leroy Sane (Schalke 04), Bastian Schweinsteiger (Manchester United/ENG), Julian Weigl (Borussia Dortmund)
Forwards: Mario Gomez (Fiorentina/ITA), Thomas Mueller (Bayern Munich), Lukas Podolski (Galatasaray/TUR), Mario Goetze (Bayern Munich), Andre Schürrle (Wolfsburg)