The Belgian football star sustained a foot injury in Real Madrid's Champions League match against Paris Saint-Germain last week and new tests show that it is a fracture of the right ankle, Real writes on his website.
Hazard is now expected to be gone for around four to six weeks.
Also, teammate Marcelo looks to miss the big game at Camp Nou as the outer back is pulled with a calf injury.
The meeting with Barcelona would have been 28-year-old Hazard's first El Clásico in his career since joining Real Madrid from Chelsea for € 100 million, equivalent to just over a billion euros, this summer.
Barcelona tops La Liga by 31 points, the same points as Real Madrid but with better goal difference.