Mukesh Dhirubhai Ambani was born on 19 April 1957 to Dhirubhai Ambani and Kokilaben Ambani. He has a younger brother, Anil Ambani, and two sisters, Dipti Salgaoncar and Nina Kothari. The Ambani family lived in a modest two-bedroom apartment in Bhuleshwar, Mumbai until the 1970s. Dhirubhai later purchased a 14-floor apartment block called 'Sea Wind' in Colaba, where, until recently, Mukesh and Anil lived with their families on different floors.

In 1980, the Indian government under Indira Gandhi opened PFY (polyester filament yarn) Manufacturing to the private sector. Dhirubhai Ambani applied for a license to set up a PFY Manufacturing plant. In spite of stiff competition from Tatas, Birlas and 43 others, Dhirubhai was awarded the licence. To help him build the PFY plant, Dhirubhai pulled his eldest son Mukesh out of Stanford where he was studying for his MBA. Mukesh Ambani then continued to work for Reliance and did not return to his university program after that. He led Reliance's backward integration from textiles into polyester fibres and further into petrochemicals, beginning in 1981.

Ambani directed and led the creation of the world's largest grassroots petroleum refinery at Jamnagar, India, which had the capacity to produce 660,000 barrels per day (33 million tonnes per year) in 2010, integrated with petrochemicals, power generation, port and related infrastructure.

During the fiscal year ending 31 March 2012, Mukesh reportedly, decided to forgo nearly ₹240 million from his annual pay as chief of Reliance Industries Ltd (RIL). He elected to do this even as RIL's total remuneration packages to its top management personnel increased during that fiscal year. This move kept his salary capped at ₹150 million for the fourth year in a row.

In December 2013 Ambani announced, at the Progressive Punjab Summit in Mohali, the possibility of a "collaborative venture" with Bharti Airtel in setting up digital infrastructure for the 4G network in India.

On 18 June 2014, Mukesh Ambani, addressing the 40th AGM of Reliance Industries, said it will invest Rs 1.8 trillion (short scale) across businesses in the next three years and launch 4G broadband services in 2015.

In February 2016, Mukesh Ambani-led Jio launched its own 4G smartphone brand named LYF. In June 2016, it was India's third-largest-selling mobile phone brand.. During the 40th annual general meeting of RIL, he announced bonus shares in the ratio of 1:1 which is the country’s largest bonus issue in India, and announced the Jio Phone at an effective price of ₹0.

Mukesh is married to Nita Ambani and has two sons, Anant and Akash, and a daughter, Isha. They live in a private 27-storey building in Mumbai named Antilia valued at US$1 billion and it is said to be one of the most expensive homes ever built. In an interview with Rajdeep Sardesai in March 2017, Mukesh said that his favourite food continued to be idli sambar and his favourite restaurant remains Mysore Café, a restaurant in Kings circle (Mumbai) where he used to eat as a student at UDCT from 1974 to 1979.

As of February, 2018, Bloomberg's "Robin Hood Index" estimated that Ambani's personal wealth was enough to fund the operations of the Indian federal government for 20 days.