Ghazala Khan, mother of Us Captain Humayub Khan, claimed that Mr. Trump was ignorant about Islam and that he did not know the meaning of the word sacrifice.
Her husband Khizr Khan also attacked Trump in a heartfelt speech to the Democratic National Convention on Thursday.
The couple's son, US Army Capt Humayun Khan, was killed by a car bomb in 2004 when he was 27 years old.
On Monday, family members of eleven service-members killed in action wrote to Mr Trump, expressing dismay at his treatment of the family of Humayun Khan, and demanding an apology.
In a heartfelt letter, she stated that "When you question a mother's pain, by implying that her religion, not her grief, kept her from addressing an arena of people, you are attacking us. When you say your job building buildings is akin to our sacrifice, you are attacking our sacrifice, this goes beyond politics. It is about a sense of decency. That kind decency you mock as 'political correctness',"
Muslims amount to around 27% of the population of US, in statements released in the Republican camp, Senate Majority Leader Mitch McConnell and House Speaker Paul Ryan condemned any criticism of Muslim Americans who serve their country.
Both also rejected the idea of a Muslim travel ban, an idea proposed the Republican presidential candidate earlier in the campaign.
But neither statement mentioned Mr Trump by name or criticised him.
Earlier, Republican Ohio Governor John Kasich, a former rival to Mr Trump for the nomination, tweeted: "Shame on him. He has no decency, he has a dark heart."