Gatwick Airport was forced to close its runway for an hour on Friday evening after a confirmed drone sighting.
Flights resumed after officials were reassured that the military measures in place meant it was still safe for planes to take-off and land.
The new sighting occurred at around 5.10pm – around 19 hours after the last known activity at 10pm on Thursday.
Monitoring service FlightRadar24 showed several flights circling south of the airport while others were diverted to land elsewhere.
“Flights have now resumed at Gatwick following a reported drone in the area,” said a spokeswoman for the airport.
“While we investigated, airfield movements were suspended. This was a precautionary measure as safety remains our main priority.
“The military measures we have in place at the airport have provided us with the reassurance necessary that it is safe to reopen our airfield.”
The closure came less than 12 hours after the airport reopened following a shutdown lasting from Wednesday evening to Friday morning triggered by drone activity over the airfield.
Sussex Police believe more than one unmanned aircraft are responsible and are investigating the possibility of multiple culprits
Assistant Chief Constable Steve Barry said there is no evidence of involvement of a foreign power or terrorism, but described the drone activity as “really high-end criminal behaviour”.
“This is a really significant criminal offence,” he added. ”There are resources here at Gatwick Airport now to mitigate the threat of that and a lot of resources to bring the offender to justice.”
Military equipment was being used to stop further disruption, with reports that the Israeli-developed Drone Dome system was being used to detect the machines using radar. It can also jam communications between the drone and its operator, enabling authorities to take control of and land the drone.
The police said they were also considering less sophisticated options such as using shotguns to blast drones out of the sky.
Transport secretary Chris Grayling said the drone activity at Gatwick was “unprecedented anywhere in the world.”
The disruption affected at least 120,000 people on Wednesday and Thursday and thousands more had already been affected on Friday before flights were suspended again.
Many travellers were forced to sleep on the airport floor as they searched for alternative routes to holidays and Christmas family gatherings.
Flying drones within one kilometre of an airport boundary is punishable with a maximum prison sentence of five years.