Legacy blood is the blood of a Man of Letters or one of their legacies.
In 1956, Cuthbert Sinclair created the Werther Box, a device intended to protect Nadia's Codex. The Werther Box could only be disarmed by all the blood of a Man of Letters or one of their legacies, but Cuthbert refused to share this with the Men of Letters and was expelled for it. One of the other Men of Letters, while trying to figure out how to disarm the Werther Box apparently figured this out or came close as he slit his own wrists and Cuthbert mentioned that he was close with his method.
In order to get Nadia's Codex to translate the Book of the Damned, Sam worked to disarm the Werther Box. With the help of a hallucination of Rowena, Sam figured out that they needed the blood of a Man of Letters or a legacy. As a legacy, Sam began spilling his blood into a collector on the box and it started to disarm, but Sam quickly realized it needed all of his blood. After finding Sam and stopping him, Dean decided to add his own blood to the mix and the blood of two legacies was enough to safely disarm the Werther Box.
- Technically anytime the Winchesters have bled over the course of the series Legacy blood has made an appearance.