Waving both Spanish and Catalan flags and cheering Spain's king, the demonstrators made their way along the elegant Passeig de Gracia, home to some of the city's most luxurious hotels, to the central Placa de Catalunya square.
Organizers said some 300,000 people took part in the demonstration. Municipal police put turnout at 65,000 people, Presstv Reported.
"It is not so much about asserting of feelings of being Spanish. We came to demand unity, the unity of Spain and Catalan society which is more and more divided," Cristian Rodriguez, a 21-year-old student, told AFP.
Polls and recent elections show the Catalonia's 7.5 million residents are roughly equally divided by the secession question.
While supporters of independence have staged massive rallies in recent years, those who want the wealthy northeastern region to remain a part of Spain have remained largely silent.
But that changed after Catalonia's regional government in October 2017 pressed ahead with a banned referendum on secession and the regional parliament then declared the region's independence to no effect.
Spain's central government responded by sacking the region's government, led at the time by Carles Puigdemont, dissolving its parliament and calling early regional elections.
Sixteen Catalan separatist leaders, including Puigdemont who is in self-imposed exile in Belgium, are facing trial over their role in the separatist push. The trial is expected to start in early 2019.
"I ask that the judges who will have to issue sentences soon not be intimidated," said Javier Megino, the vice president of the group which organized the demonstration, "Spain and Catalonia", in an address to the crowd.
"Puigdemont to prison," the crowd chanted in response.
Puigdemont was replaced by Quim Torra as Catalonia president following snap polls in the region in December 2017 which saw separatist parties once again win an absolute majority in the regional parliament.
Torra's government is divided between those who back disobedience to advance the cause of independence and those who favor dialogue with new Spanish Prime Minister Pedro Sanchez.
In power since June, Sanchez has taken a softer line on Catalonia than his conservative predecessor, a stance which many at the demonstration criticized.
Esther, a 63-year-old pensioner who declined to give her last name, said the central government was making concessions to the Catalan government which was "giving wings to the separatists".
October 12 is known as Dia de la Hispanidad, or Hispanic Day, and celebrates Christopher Columbus' arrival in the New World. The day was declared Spain's national day in 1987.