He was born in Saint Malo and, until recently, was thought to have joined the French East India Company at the age of 11 as a sub-lieutenant aboard the Duc de Bourgogne. However, the Australian historian Edward Duyker, in the (revised) French edition of his biography of Marion Dufresne, has revealed that this was in fact the future explorer's elder brother.

During the War of the Austrian Succession, he commanded several ships and was a captain by 1745. In the Seven Years' War, he was engaged in various naval operations. After the war, he again sailed on the East India routes and eventually settled in Port Louis on Mauritius, where he also was the harbourmaster for some time.

When the French East India Company collapsed and was dissolved in 1769, du Fresne was suddenly unemployed. He convinced Pierre Poivre, the civil administrator, to equip him with two ships and send him on a twofold mission: first, he was to bring Ahu-toru, a Tahitian who had been brought to Paris and displayed there, but brought back only as far as Mauritius, back to Tahiti; and second, he was to search for the southern continent. Du Fresne was given two ships, the Mascarin and the Marquis de Castries. Ahu-toru died of smallpox shortly after their departure from Port Louis.

On this expedition, he discovered first the Prince Edward Islands and then the Crozet Islands before sailing towards Australia. They spent a few days in Tasmania, where Marion Bay in the south-east is named after him. He was the first European to explore the island and, due to his interaction with the Tasmanian Aboriginies, was the first person to show that Australia was not terra nullius.

He sighted New Zealand's Mount Taranaki on March 25, 1772, and named the mountain Pic Mascarin without knowing that James Cook had named it "Mount Egmont" three years earlier.

Over the next month, they explored the islands, repaired their ships and treated their scurvy, first anchoring at Anchor Cove and later in the Bay of Islands. Apparently their relations with the Māori were peaceful at first; they could communicate thanks to their Tahitian vocabulary learned from Ahu-toru, and the Māori even held a ceremony for them.

However, the French appear to have broken tapu by fishing in Manawaora Bay. Tapu had been placed on the area after members of the local tribe drowned here some time earlier, and their bodies had been washed up at Tacoury's Cove. The local Māori believed that the violation would anger not only the gods but neighbouring tribes, provoking war. So on July 12, 1772, a few hundred Māori warriors set on du Fresne and his fishing crew, who had unsuspectingly arrived in his favourite fishing area in a small "gig". Du Fresne and twenty six men of his crew were killed and eaten, including de Vaudricourt and Lehoux, Pierre (a volunteer), Thomas Ballu of Vannes, Pierre Mauclair (the second pilot) from St Malo, Louis Ménager (the steersman) from Lorient, Vincent Kerneur of Port-Louis, Marc Le Garff from Lorient, Marc Le Corre of Auray, Jean Mestique of Pluvigner, Pierre Cailloche of Languidic and Mathurin Daumalin of Hillion.

Lieutenant Crozet went with a small party in search of du Fresne when he did not return to the Mascarin. When Crozet learned of their deaths, he returned to the ship. In retaliation, the French burned down a village named Paeroa, killing 250 Māori. They named the bay "Anse des Assassinats" (Assassination Cove).

They left on 12 July 1772. The French buried a bottle at Waipoa on Moturua, containing the arms of France and a formal statement taking possession of the whole country, with the name of "France Australe."