"The Scottish people voted decisively to keep our United Kingdom together, a result which both the Scottish and U.K. governments committed to respect," Johnson wrote in a letter Tuesday to Scottish First Minister Nicola Sturgeon, who requested the second vote.
Johnson reminded Sturgeon that the 2014 referendum -- in which more than 55 percent of Scots favored being part of the U.K. -- was a "once in a generation" vote.
"Another independence referendum would continue the political stagnation that Scotland has seen for the last decade, with Scottish schools, hospitals and jobs again left behind because of a campaign to separate the U.K.," he wrote.
Sturgeon, leader of the pro-independence Scottish National Party, cited uncertainty surrounding Great Britain's departure from the European Union as a reason for the push.
She said that most Scots voted against Brexit in 2016.
“If we are to safeguard Scotland’s interests, we cannot wait indefinitely," Sturgeon told Scottish lawmakers in Holyrood last year. "That is why I consider that a choice between Brexit and a future for Scotland as an independent European nation should be offered in the lifetime of this Parliament."