Ms Chernukhin gave another £80,000 in donations in the final months of 2021, sums reported to the electoral watchdog by the Tories in February.
The British citizen has donated more than £2m to the Conservative HQ, local Tory parties and Tory MPs over the past eight years, as well as paying to play tennis with Mr Johnson at a fundraising auction.
Labour MP Bill Esterson asked Mr Johnson at PMQs on Wednesday whether he would ask the Tory party to give Ms Chernukhin’s donations to “Ukrainian humanitarian causes”.
Dismissing the request, the prime minister said: “It’s absolutely vital that if we are to have a successful outcome in what we are trying doing … that we demonstrate that this is not about the Russia people – this is about the Putin regime.”
Foreign Office minister James Cleverly recently defended donations by Ms Chernukhin, saying: “She has every right to donate to us or any other political party.”
The senior figure told LBC all Tory donors “are registered, are legitimate” and suggested Russia-linked donors “support our aims or objectives.”
Asked on BBC Breakfast if she was embarrassed to have attended a diner with Ms Chernukhin, foreign secretary Liz Truss replied: “No I am not.”
Labour has called on the Tories to hand back Russia-linked donations, estimating that almost £1.9m has been given by such individuals either to the party HQ or Tory MPs since Mr Johnson entered No 10.
The latest Electoral Commission figures show £4.9m was donated to the Tories in the final quarter of last year, a marked increase from the £3.7m received by the party in the previous quarter.
Labour has warned ministers that the government was “taking too long” to bring in sanctions against Russian oligarchs.
Chris Bryant MP suggested in the Commons that Chelsea FC owner Roman Abramovich was “terrified” of facing such measures – and claimed the billionaire was considering selling his home in London.
Sir Keir Starmer also used PMQs on Wednesday to demand to know why Mr Abramovich has not yet been sanctioned.
The Labour leader also pointed out that a register of the real foreign owners of property will not come into force until autumn 2023 at the earliest – “far too long for the Ukrainian people”.
Speaking at PMQs, Tory MP Bob Seely claimed UK lawyers were working on behalf of Russian oligarchs – accusing them of putting pressure on British media organisations.
Mr Johnson replied: “The legal profession – everybody involved in assisting those who wish to hide money, assisting corrupt oligarchs – have been set on notice that their actions are under scrutiny.”
Ms Truss told MPs last week that British government moves to sanction Russian oligarchs was being slowed down by law firms based in London.
Officials have to draw up detailed cases for action against each individual and business, with the aim of making the justification watertight in the case of a court challenge.
In a possible hint that the government could still go further in terms of individual sanctions, Mr Johnson said the silence of Russian oligarchs who had investments in the UK over the Ukraine invasion was “inexplicable”.
The prime minister pressed them to “denounce this act of aggression”.