The Brazilian-born midfielder obtained Portuguese citizenship in December 2013 and, despite previously stating he would only ever consider representing Brazil at international level, began proceedings to be given clearance to play for the Seleccao.
Portugal had hoped that Diego Costa's accepted decision to represent Spain over Brazil would be a suitable precedent to allow Fernando's request to be granted, but Fifa has since made it clear the 26-year-old can only play for the country of his birth as he represented the Under-20 side in World Cup qualifying in 2007.
A statement released by the Portuguese FA on Friday said: "The Portuguese Football Federation was informed this Friday by Fifa that the player Fernando Reges isn't elibigle to represent the national team.
"Fifa considered that the player did not hold Portuguese nationality when he played for Brazil in the U20 World Cup qualifiers, as required by federation regulations."
Fernando, who has made 38 appearances in all competitions for Porto this season, has long been linked with a possible move to Premier League giants Manchester City.