The organisation, Lakshya Parivar, broke the previous record set at California in the USA, where blood tests were conducted on 1,460 people in six hours, Asia head of Golden Book of World Records, Manish Vishnoi, said.
Lakshya Parivar set the record on Wednesday.
"In a period of six hours, Lakshya Parivar conducted blood tests on 2,501 people," Mr Vishnoi said.
Mr Vishnoi presented the certificate of the world record to Chandan Sharma, convener of the organisation, yesterday.
The Golden Book of World Records team recorded the entire process on cameras.
According to Chandan Sharma, his organisation chose the Valentine's Day to set the record to send a positive message to the society.
"Apart from setting the record, 260 people also donated blood for thalassemia patients. We chose the Valentine's Day to set the record to send a positive message among the youths. We informed about the blood group to the people, who took part in the record feat," Sharma said.