75-year-old Ansari, a former IFS officer, got 490 votes against 238 bagged by Mr Singh out of 736 votes polled earlier in the day.
Altogether 787 Members of Rajya Sabha and Lok Sabha were eligible to vote but 47 MPs did not cast their votes, who include ailing Union Minister Vilasrao Deshmukh who has been admitted in a Chennai Hospital.
Eight votes were invalid, according to T K Vishwanathan, Lok Sabha Secretary General and Returning Officer for the Vice-Presidential poll.
Mr Ansari is the second Vice-President to be re-elected after S Radhakrishnan who was the first to get two terms from 1952 to 1962.
An affable and well-read person, Mr Ansari was the second choice of Congress president Sonia Gandhi for the President's post and lost out to Pranab Mukherjee due to some brinkmanship by TMC leader Mamata Banerjee and Samajwadi Party leader Mulayam Singh Yadav.
Formerly the Vice-Chancellor of Aligarh Muslim University, Mr Ansari's name came as a surprise in 2007 election when the Left parties, supporting the UPA-I from outside, proposed him and the Congress-led alliance accepted it.
The Left parties, which faced divisions on the Presidential poll, however, had no problems in supporting Mr Ansari for a second time.