World Health Organisation officials have traced an Ebola-like disease to the town of Yambio, in the Western Equatorial region of the African country.
Fifteen cases of the potentially fatal virus have been confirmed over the past fortnight and four people have died.
More than 100 others are being kept under surveillance.
Four people are in isolation at Yambio Hospital, so the total number of infected cases could still rise.
The Ebola virus in its early stages is hard to diagnose because some of the symptoms, such as fever and joint pain, mimic malaria.
Many people go on to develop internal bleeding, a characteristic typical of Ebola, and without medical attention the prospects are bleak.
A crisis group has been set up with aid agencies in the region, in an attempt to curtail the spread.
The last major outbreak was in Uganda two years ago; hundreds died. The hope is that quick action now will avert a similar crisis in southern Sudan.