Massimino was born August 19, 1962 in Oceanside, New York, and raised in Franklin Square, New York, both on Long Island. He graduated from H. Frank Carey Junior-Senior High School in Franklin Square, New York in 1980. He went on to attend Columbia University, graduating with a Bachelor of Science degree in Industrial Engineering in 1984. He then attended the Massachusetts Institute of Technology, graduating with a Master of Science degree in Mechanical Engineering and a Master of Science degree in Technology and Public Policy in 1988. He continued his education at MIT, earning a Degree of Mechanical Engineer in 1990 and a Doctor of Philosophy degree in Mechanical Engineering in 1992.

Upon completing his B.S. degree from Columbia, Massimino worked for IBM as a systems Engineer in New York City from 1984 until 1986. In 1986 he entered graduate school at the Massachusetts Institute of Technology where he conducted research on human operator control of space robotics systems in the MIT Mechanical Engineering Department's Human-machine systems Laboratory. His work resulted in the awarding of two patents. While a student at MIT he worked during the summer of 1987 as a general Engineer at NASA Headquarters in the Office of Aeronautics and Space Technology, during the summers of 1988 and 1989 as a research fellow in the Man-Systems Integration Branch at the NASA Marshall Space FLIGHT Center, and during the summer of 1990 as a visiting research Engineer at the German Aerospace Center (DLR) in Oberpfaffenhofen, Germany. After graduating from MIT in 1992, Massimino worked at McDonnell Douglas Aerospace in Houston, Texas as a research Engineer where he developed laptop computer displays to assist operators of the Space Shuttle remote manipulator system. These displays included the Manipulator Position Display, which was evaluated on STS-69. From 1992 to 1995 he was also an adjunct assistant professor in the Mechanical Engineering & Material Sciences Department at Rice University, where he taught feedback control of mechanical systems. In September 1995, Massimino joined the faculty of the Georgia Institute of Technology as an assistant professor in the School of Industrial and Systems Engineering. At Georgia Tech he taught human-machine systems engineering classes and conducted research on human-machine interfaces for space and aircraft systems in the Center for Human-Machine Systems Research, and is currently an Adjunct Assistant Professor at Georgia Tech. He has published papers in technical journals and in the proceedings of technical conferences. In 2011, Massimino co-hosted season three of National Geographic Channel's Known Universe documentary series along with Sigrid Close, Andy Howell, David E. Kaplan, and Steve Jacobs.

Massimino married his wife Carola on August 11, 1989 and they have two children: daughter Gabby (Gabriella) and son Daniel.

Selected as an Astronaut candidate by NASA in May 1996, Massimino reported to the Johnson Space Center in August 1996. He completed two years of initial training and evaluation and qualified for FLIGHT assignment as a mission specialist. Prior to his first space FLIGHT assignment, Massimino served in the Astronaut Office Robotics Branch, and in the Astronaut Office Extravehicular Activity (EVA) Branch. He has served as a CAPCOM (spacecraft communicator) in Mission Control and performed other technical tasks in the Astronaut Office CAPCOM Branch. Currently he is on loan from the Johnson Space Center and serves as a visiting professor at Columbia University teaching a course on human spaceflight.

STS-125 Atlantis (May 11–24, 2009) was the final Hubble servicing mission by the Space Shuttle. Atlantis landed in California after stormy weather prevented the shuttle from landing at NASA's home base in Florida as previously planned. During the mission Massimino became the first person to use Twitter in space, writing, "From orbit: Launch was awesome!! I am feeling great, working hard, & enjoying the magnificent views, the adventure of a lifetime has begun!" Rather than using the Service directly while in orbit, Massimino composed his messages and shared them with Mission Control which then posted the messages via the Internet to Twitter. Massimino's public updates about his training for the STS-125 mission began April 3, 2009.

Massimino makes a brief appearance at a shuttle launch in Chapter XXIX of the 2012 novel A Hologram for the King, in which he discusses his experience in space.

After the October 4, 2013 release of the movie Gravity with Sandra Bullock, Massimino commented on the scientific reliability of the movie. It was also suggested that the role played by George Clooney was partially based on Massimino's spacewalk performance.

On July 29, 2014, Massimino announced that he would be leaving NASA to pursue a teaching career at Columbia University, in New York City.

Massimino appeared in a 2015 MasterCard Rewards commercial.

Massimino's book Spaceman: An Astronaut's Unlikely Journey to Unlock the Secrets of the Universe was published by Crown Archetype on 4 October 2016. The Audible e-book version is narrated by Massimino himself.

In 2017, Massimino hosted Science Channel's live coverage of the total solar eclipse of August 21, dubbed the "Great American Eclipse."

Soon after, he became host of Science's The Planets and Beyond, an in-depth look at the planets of the Solar System as well as dwarf planets and exoplanets.. The series was renewed for a second season, which began airing in 2018. He also hosted an online companion series, Ask the Astronaut, in which he talked about his personal experiences of spaceflight.