The rocket, whose inaugural mission had been delayed by several years, blasted of at 2:03 am (0703 GMT) from the Cape Canaveral Space Force Base in the US state of Florida, the webcast showed.
The mission is seen as critical to Blue Origin's efforts to compete with Elon Musk's SpaceX, which dominates the commercial space industry.
"LIFTOFF! New Glenn is beginning its first ever ascent toward the stars," Blue Origin said on social media platform X.
"New Glenn has passed the Karman line, the internationally recognized boundary of space!" the firm posted just a few minutes later.
And then: "Second stage engine cutoff confirmed. New Glenn's second stage and payload are now in orbit."
An initial test launch of the towering 320-foot (98-meter) rocket, dubbed New Glenn in honor of legendary American astronaut John Glenn, had been scrubbed early Monday morning after repeated halts during the countdown.
The company later said it had discovered an icing issue on a purge line and would aim for a possible early Tuesday morning launch, but that weather conditions were unfavorable.
On Monday night, Blue Origin announced that launch had been postponed.
With the mission, dubbed NG-1, Amazon founder Bezos is taking aim at the only man in the world wealthier than him: fellow tech innovator Musk.
Musk's company SpaceX dominates the orbital launch market through its prolific Falcon 9 rockets, which have become vital for the commercial sector, Pentagon and NASA.
"SpaceX has for the past several years been pretty much the only game in town, and so having a competitor... this is great," G. Scott Hubbard, a retired senior NASA official, earlier told AFP, expecting the competition to drive down costs.
Upping the high-stakes rivalry, SpaceX also plans another orbital test this week of Starship -- its gargantuan new-generation rocket.
- Landing attempt -
Blue Origin will now attempt to land New Glenn's first-stage booster on a drone ship stationed about 620 miles (1,000 kilometers) downrange in the Atlantic Ocean.
SpaceX has made such landings now routine, but this will be Blue Origin's first shot at the sci-fi feat.
High seas last week caused the New Glenn launch to be pushed back several days.
Meanwhile, the rocket's upper stage will fire its engines toward Earth orbit, reaching a maximum altitude of roughly 12,000 miles above the surface.
A Defense Department-funded prototype of an advanced spaceship called Blue Ring, which could one day journey through the solar system, will remain aboard for the roughly six-hour test flight.
Blue Origin has experience landing its New Shepard rockets -- used for suborbital tourism -- but they are five times smaller and land on terra firma rather than a ship at sea.
Physically, the gleaming white New Glenn dwarfs SpaceX's 230-foot Falcon 9 and is designed for heavier payloads.
It slots between Falcon 9 and its big sibling, Falcon Heavy, in terms of mass capacity but holds an edge with its wider payload fairing, capable of carrying the equivalent of 20 moving trucks.
- Slow v fast development -
Blue Origin has already secured a NASA contract to launch two Mars probes aboard New Glenn. The rocket will also support the deployment of Project Kuiper, a satellite internet constellation designed to compete with Starlink.
For now, however, SpaceX maintains a commanding lead, while other rivals -- United Launch Alliance, Arianespace, and Rocket Lab -- trail far behind.
Like Musk, Bezos has a lifelong passion for space.
But where Musk dreams of colonizing Mars, Bezos envisions shifting heavy industry off-planet onto floating space platforms in order to preserve Earth, "humanity's blue origin."
If New Glenn succeeds, it will provide the US government "dissimilar redundancy" -- valuable backup if one system fails, said Scott Pace, a space policy analyst at George Washington University.
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