Commons standards commissioner Kathryn Stone has confirmed she is looking into whether the prime minister correctly declared how the trip was paid for.
Mr Johnson has previously declared he received accommodation worth £15,000, covered by Carphone Warehouse co-founder David Ross.
Downing Street said the prime minister had acted correctly and transparently.
But Labour said there were “questions” over Mr Ross’s “generosity”.
The commissioner normally investigates whether MPs have failed to declare a financial interest, not declared it correctly, or declared it late.
Labour calls for probe into Johnson’s £15,000 holiday
What are the rules for MPs on financial interests?
Mr Ross initially said he had not paid “any monies” for the trip Mr Johnson took with his partner Carrie Symonds to the private island of Mustique – part of St Vincent and the Grenadines – between Boxing Day 2019 and 5 January 2020.
He later clarified that he had “facilitated” accommodation for the prime minister.
Mr Ross added that Mr Johnson’s declaration of a “benefit in kind” in the February 2020 MPs’ register of MPs’ interests had been “correct”.
But Labour asked for the standards commissioner to investigate, saying Mr Johnson’s entry appeared “to be incorrect”.
On Monday, Ms Stone confirmed that the prime minister was among nine MPs currently being investigated for potential breaches of the MPs’ code of conduct.
She is examining whether Mr Johnson followed Commons rules on declaring financial interests relating to trips outside the UK.
These state that MPs have to register visits costing more than £300, where these have not been wholly covered by MPs themselves or the taxpayer.
Ms Stone will report her findings to the Commons Standards Committee, which can order MPs to apologise or pay back money if they are found in breach of the code.
In extreme cases it can recommend MPs are suspended, although this requires approval from the whole Commons and has only happened a handful of times.
Labour deputy leader Angela Rayner said: “The public have a right to know who paid for Boris Johnson’s luxury Caribbean holiday and the renovation of his flat. Most importantly, we need to know what these donors were promised or expected in return for their generosity.”
But a Downing Street spokesman said: “The prime minister transparently declared the benefit in kind in the Commons register of interests.”
He added: “Clearly the rules are set out and it’s important that everyone abides by them, as the prime minister has done throughout.”
Labour has also asked Ms Stone to investigate the prime minister over how a renovation of his official Downing Street flat was initially paid for.
In April 2019, Mr Johnson was rebuked by the parliamentary commissioner for standards for failing to register a share of a Somerset property within 28 days of acquiring it.