Some 7.2 million Tunisians are eligible to elect a 217-member assembly that will write a new constitution after decades of autocratic government under Ben Ali, ousted in a popular revolt.
Polls opened at 07.00 am (0600 GMT), with the independent elections body (ISIE) urging voters to turn out in numbers "to continue the work of the martyrs and to realise the objectives of the Tunisian revolution".
"I am very excited about voting in the first democratic elections. Freedom was expensive, we have to pay our dues!" hotel employee Mondher Hamdi, 23, told AFP.
"I didn't sleep at all last night from excitement," Houcine Khlifi, 62, said while queuing outside a central Tunis polling station.
"Tunisia today offers to the world a bouquet of flowers of liberty and dignity."
Ben Ali was ousted in nation-wide pro-democracy uprisings which claimed their latest Arab strongman Thursday with the killing of Moamer Kadhafi of Libya, which will declare its official "liberation" Sunday as Tunisians cast their ballots.
Unlike its neighbour, which descended into civil war, Tunisia's path to democracy has been mostly peaceful apart from some protests against the pace of transformation and sporadic violent outbursts by conservative Islamists against secularisation.
The multi-party body elected Sunday will have the loaded task of appointing an interim president and a caretaker government for the duration of the constitution drafting process, expected to take about a year.
The Islamic Ennahda party, banned under Ben Ali, is polled to win the biggest bloc of votes in this first-ever open contest in a country where the outcome of elections used to be a foregone conclusion.
The constituent assembly will have to choose what type of government the country will have and how to guarantee basic liberties, including women's rights which many fear Ennahda would seek to diminish despite its assurances to the contrary.
The progressive left, however, remains divided with party leaders having failed to form a pre-vote alliance.
Elections chief Kamel Jendoubi on Saturday declared his ISIE polling commission "ready and confident", while the European Union observer mission said there was "almost no chance of cheating or falsifying results".
Ennahda had warned of a risk of vote rigging and vowed a fresh uprising if it detected fraud, but its leader Rached Ghannouchi stressed at a final rally Friday that the party would recognise the results "no matter Ennahda's score."
In what is widely regarded as the Arab Spring's first democratic test, Tunisians can choose from more than 11,000 candidates -- half of them women -- representing 80 political parties and several thousand independents.
Vote counting will start as soon as polling stations close at 07.00 pm, with results updated live throughout the night.
The final tally will be released on Monday.