The firm sparked outrage after sacking 800 seafarers without notice on March 17, amid plans to bring in cheaper agency staff.
P&O boss Peter Hebblethwaite will appear before a Scottish Parliament committee on Tuesday, after he was heavily criticised over the job cuts.
The MCA said it was “in the process” of holding The Pride of Kent on Monday.
A spokesperson for the MCA said: “Our surveyors are in the process of detaining the Pride of Kent. We are awaiting confirmation of all the detainable items.”
Transport Secretary Grant Shapps confirmed the news in a tweet, adding that the ship was being held following an inspection and that “safety will not be compromised”.
It follows the detention of another P&O vessel which was held in the Northern Ireland port of Larne on Friday due to “failures on crew familiarisation, vessel documentation and crew training”.
The Rail, Maritime and Transport union said it believed the MCA acted because of “multiple safety and operational breaches”, including the wearing of breathing apparatus.
The union repeated its demand that the Government seize P&O’s entire fleet and take action to get them back in service with the sacked crew reinstated.
General secretary Mick Lynch said: “The seizing of the Pride of Kent by the MCA this evening should be adequate evidence for the Government that the gangster capitalist outfit P&O are not fit and proper to run a safe service after the jobs massacre.
“It’s rare enough for the MCA to impound a ferry but P&O have now had two in a week after the jobs carve up which speaks volumes about the dire state of their operation.
“It’s now high time for these important vessels to be taken over under public control with the sacked crews reinstated as the only way to get these crucial ferry routes back running safely.”
The MCA said another of the firm’s vessels, European Causeway, remains under detention in Larne.
Mr Shapps wrote to Mr Hebblethwaite earlier on Monday threatening to bring a “package of measures” to Parliament to block the company from paying below minimum wage.
“Through that package, I intend to block the outcome that P&O Ferries has pursued, including paying workers less than the minimum wage,” he said.
The measures could be unveiled on Wednesday.
The minimum wage in the UK for people aged 23 and above is £8.91 per hour.
Mr Hebblethwaite, whose basic annual salary is £325,000, told MPs on March 24 the average pay of the agency crew is £5.50 per hour.
He will face the Net Zero, Energy and Transport Committee on Tuesday after he appeared before a Westminster committee last week.
Irish Ferries began operating on the Dover to Calais route in June 2021 in competition with P&O Ferries.
Irish Ferries did not immediately respond to a request for a comment on Monday.
A P&O Ferries spokesman said: “We fully welcome the Government’s commitment to increasing the minimum wage for all seafarers working in British waters.
“From the outset, we have called for a level playing field when it comes to pay and conditions on British ferry routes.
“Our announcement is not about reducing seafarer’s wages, it is to enable us to have a fully flexible crewing model that allows us to meet the demands of our customers.
“The predicted savings we announced are not solely coming from the reduction in wages, but from removing job duplication and the benefits we will see from increased flexibility.”