Nearly 400,000 people have signed up to join the demonstration in Barcelona, part of an escalating struggle between the wealthy northeastern province and Spain's central government.
The protest coincides with Catalonia's national day, the "Diada," which commemorates the fall of Barcelona in the War of the Spanish Succession in 1714 and the region's subsequent loss of institutions and freedoms.
Since 2012 the holiday has been used by separatists to press for an independent state -- but Catalonia's pro-separatist president Carles Puigdemont said this year's "Diada" was "very important".
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"It is a day to express our will as a people, remembering the past, where we come from, but also to project ourselves into the future," he said in a speech broadcast on Catalan TV on the eve of the march.
"A future that we have in our hands and that we will democratically decide really soon."
Organisers have chartered some 1,800 buses to take people to Barcelona for the march, which gets underway at 17:14 (1514 GMT) -- a nod to the year 1714.
The demonstrators will take the shape of a giant "X" by gathering on the Paseo de Gracia and Aragon avenues in central Barcelona to represent the mark Catalans will make on their ballots during an independence referendum slated for October 1st.
If the "Yes" side wins the vote, Catalonia's regional government has vowed to declare independence within 48 hours and set about building a sovereign state.
With the Spain's central government promising to block the referendum, the pro-independence camp is keen to show that it can rally its troops -- especially after participation in the Diada declined last year.
"This Diada everyone must ask themselves: what will you do on October 1?
Help the vote or help to stop it?," Catalan government spokesman Jordi Turull said Sunday.
"History has called on us and it is a great honour but also a great responsibility."
Prime Minister Mariano Rajoy's conservative government argues the referendum violates the constitution, which states that Spain is indivisible.
"There will not be a referendum," Rajoy told officials from his conservative Party Popular on Saturday.
Following a legal challenge from Rajoy's government, Spain's Constitutional Court suspended a referendum law that was fast-tracked through Catalonia's regional parliament on Wednesday.
Police have searched Catalan printing houses suspected of making ballots for the referendum while Spain's state prosecutor has launched criminal proceedings against Puigdemont and other top Catalan officials.
Catalan society is deeply divided over independence.
In a survey by the Catalan Centre of Opinion Studies in July, 41.1 percent backed independence while 49.9 percent rejected it. Some 70 percent wanted a referendum, however, to settle the question once and for all.
Like the referendum held in Britain last year on the country's membership in the European Union, the issue in Catalonia pits rural areas -- which are more pro-independence -- against large urban centres like Barcelona that are more in favour of remaining within Spain.
Catalonia, which is roughly the same size of Belgium and has its own language and customs, already has significant powers over matters such as education and healthcare.
But Spain's economic problems, coupled with a perception that the wealthy northeastern region's 7.5 million people pay more in taxes to Madrid than they get in return, have pushed the independence question to centre stage.
During the "Diada" 2013 demonstrators formed a human chain that crossed Catalonia.
The following year the demonstration took the shape of a giant "V" for "vote" down two of the main avenues of Barcelona. In 2015 the march resembled a human arrow.