NHS officials revealed on Tuesday night that the 100-acre Docklands site had started receiving patients on Monday.
Unlike during the first wave, the overflow facility is admitting non-Covid patients to create more room in the critical care wards of permanent hospitals for those with the virus.
The announcement came as the number of Covid patients in London reached 7,606, with 1,085 currently on ventilators.
Dr Vin Diwakar, NHS England's medical director for London, said that on top of huge numbers of Covid patients trusts in the capitals are currently treating three times this many people without Covid.
"As the pressure from Covid-19 has increased, this has put pressure on the number of beds that we have for other conditions," he said at a Downing Street press conference. "That's why yesterday we opened the Nightingale hospital and admitted our first patients. This time it's taking patients who do not have Covid."
Dr Diwakar warned that "we cannot do this indefinitely", saying that even with the use of the Nightingale capacity would overflow on the current trajectory.
Located at the vast ExCel convention centre in east London, the temporary hospital is also hosting one of the first NHS mass vaccination hubs, which came on stream on Monday.
It was opened to great fanfare on April 3, only 10 days after the project was announced by Matt Hancock, the Health Secretary. However, by May 8 it was placed on standby, having discharged the last of its 54 coronavirus patients.
It emerged in April that the Nightingale had only treated 19 patients over the Easter weekend because existing hospitals had increased their intensive care capacity.
The Nightingale in Manchester was reopened in October, also as a "step-down" facility for non-Covid patients.