It comes after he said the Little Scholars Nursery in Broughty Ferry, Dundee had repeatedly told his wife, Nadia El-Nakla, that there was no place available for their two-year-old daughter Amal.
But speaking to the Daily Record on Monday, the couple claimed that - just two days after their rejection - when a white friend asked if there was a space for her two-year-old son, the nursery confirmed its availability on three afternoons a week.
A spokesman for the nursery owners insisted they were “extremely proud of being open and inclusive to all” insisting that “any claim to the contrary is demonstrably false and an accusation that we would refute in the strongest possible terms”.
The spokesman said: “In addition to our owners being of Asian heritage, across more than a decade we have regularly welcomed both children and staff from a range of different religious, cultural, ethnic and racial backgrounds, including two Muslim families currently.
“We have also regularly made arrangements to accommodate different lifestyles by, for example, providing a halal menu for those children who come from Muslim families.”
Taking to Twitter after the news broke, however, Mr Yousaf hit out at the nursery’s statement, claiming that the business was given “ample time” to explain the discrepancy.
“Instead their response is effectively the ‘I cant be racist my pal is Black’ defence. That doesn't fly any more,” he added.
“At the beginning of this process I really did not think this could be Islamaphobic or racist. It was my wife's gut instinct that something just wasn't right.”
Mr Yousaf went on: “People (understandably!) see me as Health Secretary, but first & foremost I'm a father/step-father to my girls.
“I know the struggles they will face throughout their lives.
“While she is oblivious to it all,” he added, “I am beyond gutted that I couldn't protect my wee girl, aged 2, from hatred.”
The Health Secretary said he and his wife had contacted the Care Inspectorate and are also seeking legal advice on the issue.