Virgin blood is the blood of a virgin.
In What's Up, Tiger Mommy?, Mr. Vili kills Shelley, a virgin, in order to obtain her virgin remains. When Mr. Vili kills Shelley her blood splatters all over the wall. During Plutus' Auction Mr. Vili bids 5/8 of Shelley's bloody virgin remains and the finger of the frost giant Ymir for Mjölnir. Plutus accepted and Mr. Vili took the hammer which Sam later borrowed to kill Beau. However, before he could return the hammer, Sam remembered what Mr. Vili said about the 5/8 virgin. Realizing how Mr. Vili got it, Sam used Mjölnir to kill the god in retaliation.
In Rock and a Hard Place, Jody Mills discovers that they need an oak stake stained in the blood of a virgin to kill the goddess Vesta. While Sam Winchester suggests his own blood as he is a born-again virgin, Jody states her belief that they need the real thing. As a result, Sam and Jody ask chastity group member Tammy for her blood. When she refuses, Jody punches Tammy in the nose, giving her a nosebleed and collects some of her blood on a napkin. Jody is later able to use the weapon they create with the stake and blood to kill Vesta.
In Who We Are, Sam discovers the Abrogation Ritual as a possible way to end the Lockdown of the Men of Letters bunker. However, Sam discovers that the ritual requires the blood of virgins and he, Dean and Lady Toni Bevell are all not virgins. Instead, the three use the Blood Purification Ritual to "revirginize" their blood for the Abrogation Ritual. While the blood works in the ritual, Arthur Ketch added a mystical dampener to the lockdown, preventing the Abrogation Ritual from working to end the lockdown.