Dutch military police tweeted at about 19:30 (18:30 GMT) to say they were investigating a "suspicious situation".
Part of the airport - one of Europe's busiest - was then closed off as police responded to the reported threat.
But about an hour later, Air Europa announced that a pilot had accidentally triggered the alarm.
"False alarm. In the flight Amsterdam-Madrid this afternoon was activated, by mistake, a warning that triggers protocols on hijackings at the airport," the airline tweeted.
"Nothing has happened, all passengers are safe and sound waiting to fly soon. We deeply apologise."
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Shortly before their announcement, Dutch military police confirmed all passengers and staff had been safely evacuated from the Madrid-bound flight.
Images posted on social media showed parts of the airport's D-pier cordoned off to the public, with passengers waiting around for information.
Flights still landed at other parts of the airport during the disruption, but emergency services scrambled and some flights were held on the tarmac.
Roberto Carrera, 38, landed at the airport in the midst of the alert at about 19:45 local time.
"The pilot let us know an incident may have happened," he told the BBC in a phone interview.
Mr Carrera said he and other passengers on his flight from Dublin were then held on the tarmac for about an hour before they were allowed to disembark.
He saw police in the terminal but described the atmosphere in the airport as calm overall, despite the disruption.
The incident was described as a GRIP-3 situation, Dutch officials said, meaning an incident or serious event with major consequences to a local population.
Regulation documents published by the US Federal Aviation Administration (FAA) explain that pilots can use a special transponder beacon code, typing 7500, to raise an alert for unlawful interference in the case of a hijacking.
It remains unclear if this is what happened during the false alarm on Wednesday.
Amsterdam's airport is one of the busiest transport hubs in Europe, handling more than 70 million passengers a year.