The Citizens were without both players for Sunday's last-gasp 2-1 victory away against Wolverhampton Wanderers in the Premier League.
De Bruyne has missed the last six games in all competitions since sustaining a groin problem in Man City's Champions League opener against Inter Milan (0-0) just over a month ago.
Walker, meanwhile, featured for England during the international break earlier this month, but he returned to the Etihad with fitness issues.
Guardiola said after the win at Wolves that he "doesn't think" either Walker or De Bruyne would be ready to return for Wednesday's fixture, and the Catalan coach has since provided a fresh update on the fitness of his squad.
De Bruyne, Walker ruled out of Sparta Prague clash
Man City supporters were boosted by the news that De Bruyne had returned to training towards the end of last week, but Guardiola has confirmed that the 33-year-old playmaker "has to" be 100% fit before he is considered again for selection.
As for Walker, Guardiola has revealed that the 34-year-old is nursing a knee injury picked up on international duty and is unable to provide a timeframe on the right-back's return.
Providing a fitness update on his squad at a press conference on Tuesday, Guardiola said: "Well, except for Rodri and Oscar [Bobb], the other ones are in training and getting better, but guys like Kevin for example are not exactly fit."
On De Bruyne, who missed five months of last season with a torn hamstring, Guardiola said: "I cannot tell you [how long he will be out for], I don't know. (Some time away before a return?) Yeah...
"It's not a new issue but [Kevin] doesn't feel completely fine. Kevin is not 22 years old he has to be fit for his football. He doesn't feel comfortable that he can express his incredible potential at his best.
"He is training better but he doesn't feel. He said he doesn't feel good. If you don't feel good, take your time."
On Walker, the Citizens boss added: "I think he is not ready. Since being with the national team he's not been well."
Meanwhile, Guardiola has confirmed that Oscar Bobb, who suffered a leg fracture in training in August, is expected to make his comeback between January and February of next year.
Ake, Foden could feature for Man City against Sparta Prague
One player who has recently recovered from injury is Nathan Ake; Guardiola did not specifically mention the defender at his press conference, but he is expected to be named in City's matchday squad against Sparta Prague after watching on as an unused substitute against Wolves.
Man City expert Steven McInerney from Esteemed Kompany has told Sports Mole that Wednesday's fixture could be a "perfect game" for Ake to make his return, having not played for the Citizens since August.
Guardiola has suggested that Ake is set to feature in some capacity against Sparta Prague, saying: "Josko [Gvardiol] cannot play all the games, every three days, that's why we need Nathan [Ake]! Otherwise we cannot compete until the end..."
Meanwhile, Guardiola was asked by reporters about Phil Foden, who has endured a disrupted start to the new season and came on for the final 25 minutes plus stoppage time against Wolves, setting up John Stones from a corner to score a dramatic 95th-minute winner.
"He feels much better, the minutes he played in the last game for example," said Guardiola. "Phil you realise immediately when he is happy and fit and mentally in the right moment you realise he's getting better and better."
Man City are preparing for their first-ever encounter with Czech champions Sparta Prague who, like City, have collected four points from their opening two Champions League matches in the league phase this term.