The wall was uncovered during a survey of an area between the port and Portus - another ancient Roman port, both of which are about 48km from the Italian capital Rome.
Researchers had earlier thought the river Tiber formed the northern edge of Ostia, but the geophysical survey showed Ostia's city wall continued on the other side of the river.
The team, led by Professor Simon Keay from the University of Southampton and Professor Martin Millet from the University of Cambridge, found the newly discovered area enclosed three huge, previously unknown warehouses - the largest of which was the size of a football pitch.
"The presence of the warehouses along the northern bank of the river provides us with further evidence for the commercial activities that took place there in the first two centuries," Keay added.
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The researchers have been using an established technique known as magnetometry, which involves systematically and rapidly scanning the landscape with small handheld instruments in order to identify localised magnetic anomalies relating to buried ancient structures.
These are then mapped out with specialised computer software, providing images similar to aerial photographs, which can be interpreted by archaeologists.
At the southernmost side of the Isola Sacra, the geophysical survey revealed very clear evidence for the town wall of Roman Ostia, interspersed by large towers several metres thick, and running east to west for about half a kilometre.
In an area close by, known to archaeologists as the Trastevere Ostiense, the team also found very clear evidence for at least four major buildings.
"In addition, there is a massive 142 metre by 110 metre fourth building - composed of rows of columns running from north to south, but whose function is unknown," Keay said.
"Our results are of major importance for our understanding of Roman Ostia and the discoveries will lead to a major re-think of the topography of one of the iconic Roman cities in the Mediterranean," he said.