The annual report sheds light on how easy or difficult it is for local entrepreneurs to open and run their businesses, comparing 183 economies.
Despite Singapore's top spot, the city-state's lead was eroded as Hong Kong made big strides in cutting red tape in the past year, according to "Doing Business 2010", jointly conducted with the International Finance Corporation.
New Zealand, Britain, Denmark, Norway, Ireland, Australia, Canada, and the United States completed top 10.
"Hong Kong's success in offering one of the best business environments globally rests on continuous reforms despite its already established position as a leader," said Geoffrey Walton, business line Leader for the World Bank's East Asia and Pacific business climate unit.
"Hong Kong has made use of online applications to make complying with business regulations easier or implemented one-stop shops for efficient processing of construction permits," he added.
The report noted that the southern Chinese financial hub abolished a tax on diesel fuel and streamlined the legal process for commercial disputes.
But IFC spokesman Hannfried von Hindenburg said highlighting Hong Kong's reform efforts did not take away from Singapore's success.
"Singapore is number one and has been for five years running," he said.
Singapore leads the world in investor protection, ahead of New Zealand and Hong Kong, the report said.
The report's criteria includes dealing with construction permits, registering property, getting credit, paying taxes, trading across borders and closing a business.
China was ranked 79th, although it, along with Vietnam and East Timor were the three most improved countries over the past five years.
Chad was bottom of the list, with the Central African Republic second bottom and Burundi third last.
Sub-Saharan Africa and South Asia remained the most difficult places to conduct business, although three African countries were among this year's top 10 most improved nations -- Rwanda, Cape Verde and Zambia.
What the stars mean:
★ Poor ★ ★ Promising ★★★ Good ★★★★ Very good ★★★★★ Exceptional