Issuing a statement just two days before the ceremony, Ms Markle settled the disputed matter of whether her father will be walking her down the aisle.
“Sadly, my father will not be attending our wedding,” she said.
“I have always cared for my father and hope he can be given the space he needs to focus on his health,” she added.
Thomas Markle reportedly underwent heart surgery this week and was believed to be recuperating in hospital on Thursday.
Ms Markle added a note of thanks to the public who have been sharing messages of support for the couple and their families.
“I would like to thank everyone who has offered generous messages of support. Please know how much Harry and I look forward to sharing our special day with you on Saturday,” she said.
Mr Markle, 73, allegedly staged paparazzi photographs of himself in the run-up to the wedding at Windsor Castle.
After the allegations surfaced, it began to be reported that Mr Markle would no longer take part in the wedding ceremony.
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The confirmation that he will not be attending comes as the town of Windsor is taken over by rehearsals for the big event on Saturday and by royalists already taking up their places to get a good view of the soon-to-be bride and groom.
:: Sky News is broadcasting live from Windsor with all the build-up to the royal wedding, with exclusive live coverage of the wedding in UHD in a special programme from 9am to 3pm on Sky News and Sky One this Saturday.