Tsitsipas, currently ranked 11th, had criticised Apostolos's coaching style after losing 6-4, 6-4 to former Japanese world number four Kei Nishikori in the second round of the Canadian Open on Thursday.
The 2023 Australian Open and 2021 French Open finalist, said Apostolos would continue to travel with him, support him and provide assistance off the field.
"It is with a heavy heart that I inform you that my cooperation with my father as a coach has come to an end," Tsitsipas said on social media platform X.
"I prefer to keep my father in his role as a father, and only as a father."
After his defeat to the 576th-ranked Nishikori, Tsitsipas had said he was disappointed with Apostolos.
"I realised I was wrong to talk to my father the way I did," Tsitsipas said in his post.
"I have trusted my father with the coaching role for so many years and I consider our partnership a success.
"I'm not sure who will take over, and I'm not in a position to decide yet."
Tsitsipas reached the quarter-finals of the Paris 2024 Olympics, losing to eventual gold medallist Novak Djokovic.
