Roy started his political career as Youth Congress leader and became close to Mamata Banerjee who was also associated with Youth Congress. Roy was founding member of All India Trinamool Congress which was formed in January 1998 as a breakaway faction of the Indian National Congress led by Mamata Banerjee. Soon he became party's face in New Delhi and was made general secretary in 2006. In 2001 West Bengal Legislative Assembly election, he was TMC candidate from Jagatdal constituency and came in second with 56,741.
In April 2006, Roy elected to Rajya Sabha and was a leader of All India Trinamool Congress from 28 May 2009 to 20 March 2012 in Rajya Sabha. In UPA II was appointed Minister of State in the Ministry of Shipping first, then Minister of State in the Ministry of Railways when Mamata Banerjee resigned. On 11 July 2011, when Prime Minister Manmohan Singh directed Mukul Roy to visit the site of derailment of the Guwahati-Puri Express in Assam, he openly defied him. This brought a lot of displeasure among the political circles and he was shunted out of the Railways portfolio in the latest cabinet reshuffle on 12 July 2011. In 2012, he was the subject of a controversy regarding the Railway Budget. This happened after Mamata Banerjee expressed her discontent with the increase in passenger fares, and demanded the resignation of Dinesh Trivedi, Railway Minister.
After Dinesh resigned, Mukul became the Railway Minister of India and he removed the fare hike introduced by the former Railway Minister Dinesh Trivedi, to much criticism. With the exit of Trinamool from UPA coalition due to an allowance of 51% FDI in retail and aviation sector, Mukul Roy's tenure of railway minister came to an end in the month of September 2012. Roy ran a special train called "DU Gyan Uday Express" a Bengali worded statement, which took 1000 students of Delhi University to places like Ahmedabad, Mumbai, Goa and Bangalore during the summer break of 2012. An application has been submitted to the Election Commission for the formation of the new political party called Nationalist Trinamool Congress Party and Mukul aide Amitabha Majumdar has been named as the President. The head office of the party is located in Dinhata, in North Bengal. Roy and Mamata had a fall out in 2015 when his name came up in Saradha scam as well as in Narada sting operation in which TMC leaders are involved. Roy was suspended form the party for six years as met senior BJP leaders like Finance Minister Arun Jaitley and BJP general secretary Kailash Vijayvargiya. He has resigned from Trinamool Congress on 25 September 2017. After that Roy also resigned from Rajya Sabha membership on 11 October 2017. Roy formally joined the Bharatiya Janata Party on 3 November 2017. Upon joining the party, he said that he felt proud of working under Prime Minister Narendra Modi.
