The 20-year-old DJ and producer has knocked Pharrell Williams's 'Happy' from the top of the charts with the Jess Glynne-assisted track.
The song becomes Glynne's second chart-topper of 2014, following the success of Clean Bandit's 'Rather Be'.
Route 94 told OfficialCharts.com: "I didn't see it coming. I'm absolutely over the moon! I never thought 'I'm gonna write a massive Number 1' or a pop hit, or whatever, I just wrote it, and what happened just happened.
"I'll be celebrating by going out for some food with my girlfriend and then throwing a MASSIVE party, so everyone come and get involved!"
'Happy' falls to number two on this week's chart, while Clean Bandit's 'Rather Be' and Katy Perry's 'Dark Horse' are non-movers at three and four respectively.
Sam Smith completes this week's top five with his debut solo single and former chart-topper 'Money on My Mind'.
A Great Big World and Christina Aguilera are down one place to six with their collaboration 'Say Something'.
Lily Allen has scored a new entry at seven with 'Air Balloon', which follows her two Top 10 singles ('Somewhere Only We Know' and 'Hard Out Here') in December.
John Legend is up one place to number eight with 'All Of Me', while Tiësto drops three places to nine with 'Red Light'.
Meanwhile, Coldplay's 'Magic' - the first single from their upcoming sixth album Ghost Stories - is at number ten.
Nick Mulvey makes his Top 40 debut with 'Cucurucu', which is a new entry at 26.
Avicii has scored this week's highest climber with 'Addicted To You', which soars 28 places to number 36 this week.
Watch the music video for Route 94 and Jess Glynne's 'My Love' below:
Source: Official Charts Company
View the latest Official Top 100 Singles Chart