Ramos made his 43rd appearances by starting the La Liga fixture at Camp Nou, with the two sides tied atop the La Liga standings at 35 points each.
The 33-year-old defender has surpassed the previous record of 42, which was jointly held by Real Madrid legends Paco Gento and Manolo Sanchis as well as longtime Barcelona star Xavi.
Ramos made his debut in the Clasico in November 2005, finishing on the losing side at the Santiago Bernabeu as two goals from Ronaldinho and one from Samuel Eto'o saw Barcelona emerge with a 3-0 victory.
Remarkably, Ramos has played in every fixture between the two sides since that game, scoring four goals in El Clasico – all on headers.
Ramos, who also holds the all-time Real Madrid record for red cards, has been sent off in five of the 42 previous Clasicos in which he's participated.
1 - @realmadriden player Sergio Ramos will become the player with the most appearances in #ElClasico history (43), surpassing Manolo Sanchís, Francisco Gento, Xavi Hernández and Lionel Messi (42 for each one). Captain pic.twitter.com/3m6pHenIOB— OptaJose (@OptaJose) December 18, 2019
The defender's 43 appearances against Barcelona have come in four different competitions: La Liga (29 appearances), the Copa del Rey (seven), the Spanish Super Cup (six) and the Champions League (once).
Though Ramos has set the record, it's possible that he will not hold the mark for very long. That's because a current Barcelona legend is right on his tail.
Lionel Messi made his 42nd career Clasico appearance on Wednesday when he started the match and at 32 years old, he could eventually secure the record for himself.
After Messi won his record sixth Ballon d'Or earlier this month, Ramos paid homage to his longtime rival as well as his former team-mate Cristiano Ronaldo, who has won five Ballons d'Or himself.
“They could make a Golden Ball only for Cristiano and Messi, depending on the results, and another for the rest," Ramos joked to TUDN.
"In the end it would be better for football.”
On his legacy, Ramos added: “I feel proud. In the end it is something that nobody has given me.
“Behind those walls it is all about work, the sacrifice, the dedication and the constancy. Because it is not only a challenge to arrive at the top, but also to stay there."