In a speech nearly entirely devoted to criticising the SNP and making the case for the future of the United Kingdom, May said there was "no economic case" for Scottish independence.
Scotland's First Minister Nicola Sturgeon has repeatedly argued that Scotland should stay in the European Single Market, even after the rest of the United Kingdom has left.
In an interview ahead of her speech, Mrs May said: "To me, politics isn't a game". Nicola Sturgeon has warned a second referendum is now "highly likely" after the Brexit vote a year ago saw a majority of Scots vote to Remain, but the weight of votes south of the border swung the vote in favour of Remain.
In last year's Brexit vote, the majority of Scottish voters wanted to remain in the European Union, while England and Wales voted to leave.
The Scot branded the Prime Minister "arrogant" after she insisted Nicola Sturgeon and the SNP had neglected the NHS and education in repeated bids to chase independence. May spoke warmly of the union - "we are four nations, but at heart one people" - but warned that she would fight against any decentralisation of power which meant "a looser and weaker union".
"As I have made clear repeatedly, no decisions now taken by the Scottish Parliament will be removed from them", said the prime minister. "Where we have spoken the language of consensus and cooperation, theirs has been the language of Westminster diktat".
22 additional charges for Endymion crash suspect
'The driver of a truck, identified as 25-year-old Neilson Rizzuto , travelled near the Endymion parade at around 6:42 p.m. At least three people were hospitalized overnight Saturday with moderate to serious injuries, authorities said.
Signalling there was now a widening gulf between the pro-UK parties over new powers for the Scottish parliament, with Scottish Labour and the Liberal Democrats calling for a federal UK, May said the original devolution arrangements were founded on membership of the EU. "They don't want a referendum, they want the SNP government to get on with the day job".
May will attack Surgeon at her conference speech But Mrs May said: "I am confident about the future of our United Kingdom and optimistic about what we can achieve together as a country".
Mrs May will say that she is focused on "strengthening and sustaining the bonds that unite us" and that keeping Scotland in the United Kingdom is a "personal priority".
The British Prime Minister was critical of the SNP and said services such as education and health had got worse under its leadership.
The Scottish Conservative leader said the PM will not reveal her hand unless Sturgeon formally requests a Section 30 order - the same process that paved the way for the 2014 poll through the Edinburgh Agreement.
The Conservatives are urging voters in the Scottish local elections to back them to send a signal to the SNP not to hold a new referendum.