Novello was born in Fajardo, Puerto Rico. She received her B.S. degree from the University of Puerto Rico at Rio Piedras in 1965 and her M.D. degree from the University of Puerto Rico School of Medicine at San Juan in 1970. She then completed her internship and residency in nephrology at the University of Michigan Medical Center in Ann Arbor, Michigan. Novello remained at Michigan in 1973-1974 on a fellowship in the Department of Internal Medicine, and spent the following year on a fellowship in the Department of Pediatrics at Georgetown University. From 1976 to 1978, she was in private practice in pediatrics in Springfield, Virginia.
In 1978, Novello joined the Public Health Service Commissioned Corps, her first assignment being as a project officer at the National Institute of Arthritis, Metabolism and Digestive Diseases of the National Institutes of Health (NIH). She held various positions at NIH, rising to the job of Deputy Director of the National Institute of Child Health and Human Development (NICHD) in 1986. She also served as Coordinator for AIDS Research for NICHD from September, 1987. In this role, she developed a particular interest in pediatric AIDS.
During her years at NIH, Novello earned an M.P.H. degree from the Johns Hopkins School of Hygiene and Public Health in 1982. From 1976, she also held a clinical appointment in pediatrics at Georgetown University Hospital. Novello also made major contributions to the drafting and enactment of the Organ Transplantation Procurement Act of 1984 while assigned to the United States Senate Committee on Labor and Human Resources.
During her tenure as Surgeon General, Novello focused her attention on the health of women, children and minorities, as well as on underage drinking, smoking, and AIDS. She played an important role in launching the Healthy Children Ready to Learn Initiative. She was actively involved in working with other organizations to promote immunization of children and childhood injury prevention efforts. She spoke out often and forcefully about illegal underage drinking, and called upon the United States Department of Health and Human Services Inspector General to issue a series of eight reports on the subject. Novello also similarly worked to discourage illegal tobacco use by young people, and repeatedly criticized the tobacco industry for appealing to the youth market through the use of cartoon characters such as "Joe Camel." A workshop that she convened led to the emergence of a National Hispanic/Latino Health Initiative.
Novello remained in the post of Surgeon General through June 30, 1993. She then served as the United Nations Children's Fund (UNICEF) Special Representative for Health and Nutrition from 1993 to 1996. In 1996, she became Visiting Professor of Health Policy and Management at the Johns Hopkins School of Hygiene and Public Health. Dr. Novello became Commissioner of Health for the State of New York in 1999.