The club's decision came after the newspaper La Aficion published a picture on Sunday showing Romano making a finger gesture as he was leaving the pitch and facing the stands at the end of the match.
"The coaching staff's reaction towards the fans during and after yesterday's match went against the values that this institution represents and promotes, reason for which the decision was taken not to continue with the working relationship from this moment," the club said in a statement.
"This is a deeply hurtful decision but we must be consistent with our values and responsibility as a model team. Ruben reacted negatively to pressure from the fans and that's something we can't cover up," club president Alejandro Irarragorri added in comments published by Reforma newspaper.
Romano had steered Santos to second place in the Apertura championship in December and the Clausura at the end of last season.
In other Mexican championship action, Chile striker Humberto Suazo injured his shoulder during Saturday's "Northern classic" against Tigres UANL which ended 0-0.
Suazo, who played at the 2010 World Cup finals in South Africa, will miss champions Monterrey's next two matches.
The shaven-headed Chilean nicknamed "Lollipop" is joint top scorer on five goals after seven matches with America's Argentine-born former Mexico striker Matias Vuoso, who was sent off after 15 minutes in their 2-1 loss to Morelia at the Azteca.
Pumas, who have 15 points from seven matches after a 1-0 away win over San Luis, are the only unbeaten team. Toluca fell to their first defeat, 3-2 at home to promoted Necaxa.
Tigres lead Group One with 14 points, four more than Guadalajara, Monterrey and Santos, while Toluca and San Luis are equal top of Group Two with 12 each.
Pumas lead Group Three with 15 points, two more than title favourites Cruz Azul, who beat Atlas 2-1 at home on Friday.
The top two in each of the three groups and the two next best-placed teams in the overall standings after the 17-match round robin phase qualify for the quarter-finals.
