Werder Bremen staged a last-gasp escape with Papy Djilobodji's 88th-minute strike giving them a 1-0 win over visiting relegation rivals Eintracht Frankfurt.
While Werder kept their place in the Bundesliga, their opponents go into a playoff against Nuremberg, who finished third in the second division.
Stuttgart, who needed a win to have a chance at earning the playoff place, quickly fell two goals behind in the first half as one of the Bundesliga's founding clubs faced their second relegation after 1975.
Daniel Didavi's 78th-minute goal gave them a glimmer of hope but Andre Schuerrle sealed Wolfsburg's win in the final minute with his second goal of the game.
The 2007 Bundesliga champions ended up with the worst ever defensive Bundesliga record, having let in 75 goals and having also scored a record seven own goals in the process.
"It is an extremely bitter and sad day for the club," said Stuttgart coach Juergen Kramny, who took over from Alexander Zorniger in November.
"The relegation hits us very hard. We will now need some time to process all of this. We had enough chances in the past weeks to get points but failed to do so."
LEWANDOWSKI RECORD
Bayern Munich forward Robert Lewandowski took the honours as the league's top scorer with 30 goals after netting in the champions' 3-1 victory over already-relegated Hanover 96.
The Poland striker is the first player to score at least 30 goals in a season since Dieter Mueller in 1977 and became the first foreign Bundesliga top scorer to net 30 times or more as he won the title for the second time in three seasons.
Bayern's title celebrations on coach Pep Guardiola's last home game, before his move to Manchester City, were cut short after several hundred fans invaded the pitch for the traditional beer showers and trophy ceremony.
"Normally these games can be dull but it was an exciting encounter with a lot of intensity," said Guardiola, whose team face Borussia Dortmund in the German Cup final on May 21.
"Now we will enjoy the day and celebrate and then we will prepare for the Cup final."
Borussia Moenchengladbach sealed fourth place with a 2-0 win at Darmstadt 98 and will go into the Champions League playoffs.
Borussia Dortmund, 10 points behind Bayern in second place, managed a 2-2 draw at home to Cologne while Bayer Leverkusen, who secured third place weeks ago, rounded off the season with a 3-2 comeback win over visitors Ingolstadt.
Schalke 04, who announced just before their game that coach Andre Breitenreiter would be sacked at the end of the season, crushed Hoffenheim 4-1 away to clinch a Europa League spot.
Mainz 05 battled to a 0-0 draw at Hertha Berlin to finish sixth and snatch the last automatic Europa League spot from the hosts, who will go into the competition's qualifying rounds.
(Reporting by Karolos Grohmann; editing by Ken Ferris and Pritha Sarkar)
