Real have been linked with re-signing van Nistelrooy as replacement for injured Gonzalo Higuain and the Holland striker expressed his interest in returning to the Bernabeu this week.
But Hamburg boss Armin Veh is desperate to keep hold of the former PSV and Manchester United marksman and has no intention of selling the 34-year-old.
The Hamburg boss told Deutsche Presseagentur: "There is a clear decision of our club: we are not going to let Ruud van Nistelrooy leave.
"I have respect for him and his point of view, I am aware he is very keen on residing in Madrid after the end of his career. I have a huge respect for his intensions and for him as a person, but he is under contract at our club.
"He is finally in great shape right now and him leaving is a clear no-go for us."
