Bloomberg's giving to the university over the years makes him its largest-ever philanthropic benefactor, the school said in a statement.
Of the new gift, $250 million will go toward the university's work to tackle global challenges such as the preservation of water resources, while the other $100 million will provide financial aid to undergraduate students, according to the school.
When he was an undergraduate at the Baltimore university, Bloomberg paid for his tuition by taking loans and working as a parking lot attendant. His first Johns Hopkins gift was $5 in 1965, a year after he received his bachelor's degree in engineering.
"Johns Hopkins University has been an important part of my life since I first set foot on campus more than five decades ago," Bloomberg said in a statement. "Each dollar I have given has been well-spent improving the institution and, just as importantly, making its education available to students who might otherwise not be able to afford it."
Bloomberg, now 70, went on to found financial news and information company Bloomberg LP and amassed a fortune estimated by Forbes magazine at $25 billion.
Bloomberg, who was elected mayor of New York in 2001, has said he will give away his fortune before he dies and has set up Bloomberg Philanthropies to distribute gifts. Besides education, his donations have targeted public health initiatives, the environment and the arts.
Bloomberg, chairman of the university's board of trustees from 1996 to 2002, has given the school a total of $1.118 billion, it said.