Recent attempts in the US to try to charge for different levels of online access web were not "part of the internet model," he said in Edinburgh.
He warned that if the US decided to go ahead with a two-tier internet, the network would enter "a dark period".
Sir Tim was speaking at the start of a conference on the future of the web.
"What's very important from my point of view is that there is one web," he said.
"Anyone that tries to chop it into two will find that their piece looks very boring."
An equal net
The British scientist developed the web in 1989 as an academic tool to allow scientists to share data. Since then it has exploded into every area of life.
However, as it has grown, there have been increasingly diverse opinions on how it should evolve.
The World Wide Web Consortium, of which Sir Tim is the director, believes in an open model.
This is based on the concept of network neutrality, where everyone has the same level of access to the web and that all data moving around the web is treated equally.
This view is backed by companies like Microsoft and Google, who have called for legislation to be introduced to guarantee net neutrality.
The first steps towards this were taken last week when members of the US House of Representatives introduced a net neutrality bill.
But telecoms companies in the US do not agree. They would like to implement a two-tier system, where data from companies or institutions that can pay are given priority over those that cannot.
This has particularly become an issue with the transmission of TV shows over the internet, with some broadband providers wanting to charge content providers to carry the data.
The internet community believes this threatens the open model of the internet as broadband providers will become gatekeepers to the web's content.
Providers that can pay will be able to get a commercial advantage over those that cannot.
There is a fear that institutions like universities and charities would also suffer.
The web community is also worried that any charges would be passed on to the consumer.
Sir Tim said this was "not the internet model". The "right" model, as exists at the moment, was that any content provider could pay for a connection to the internet and could then put any content on to the web with no discrimination.
Speaking to reporters in Edinburgh at the WWW2006 conference, he argued this was where the great benefit of the internet lay.
"You get this tremendous serendipity where I can search the internet and come across a site that I did not set out to look for," he said.
A two-tier system would mean that people would only have full access to those portions of the internet that they paid for and that some companies would be given priority over others.
But Sir Tim was optimistic that the internet would resist attempts to fragment.
"I think it is one and will remain as one," he said.
The WWW2006 conference will run until Friday at the International Conference Centre in Edinburgh.