Paleontologists discovered the sample in the reproductive tract of an ancient female crustacean encased in resin -- one of several samples of ostracods from Myanmar.
The previously unknown species of crustacean, now named Myanmarcypris hui, resembles a modern day mussel and is an example of an ostracod.
Ostracods are small animals that date back some 500 million years, and can still be found in oceans, freshwater lakes and rivers.
Using 3D X-ray reconstructive technology, scientists analyzed several ostracod specimens, studying their limbs and reproductive organs.
Experts discovered ripe sperm inside the sperm receptacles of a female crustacean, who would have stored the sperm for release once her eggs had matured had she not been encased in the sticky tree resin.
"This female must have mated shortly before being encased in the resin," He Wang, from the Chinese Academy of Sciences in Nanjing, said in a statement.
Scientists said the discovery marks the oldest fossil in which sperm cells have been conclusively identified.
The results "double the age of the oldest unequivocal fossil animal sperm," the report authors wrote in findings published in the Royal Society journal on Wednesday.
Specimens of fossilized sperm are very rare -- according to the report authors, the oldest known ostracod sperm are 17 million years old, and the previous record age of 50 million years was held by a species of worm.
Analysis on the ostracod samples, which date from the Cretaceous period, also revealed rare details of the crustacean's internal and reproductive organs, including the male clasper, sperm pumps, hemipenes, eggs and female seminal receptacles with giant sperm.
While the majority of male animals produce large numbers of small sperm, ostracods, the report authors said, produce small numbers of oversized sperm, with long motile tails.
Scientists said that the discovery provides "unprecedented insights into an unexpectedly ancient and advanced instance of evolutionary specialization."
Researchers added that the evidence of use of giant sperm 100 million years ago is proof of a successful long term reproductive strategy.
"The complexity of the reproductive system in these specimens raises the question of whether the investment in giant sperm cells might represent an evolutionarily stable strategy," Renate Matzke-Karasz, a geobiologist at Ludwig-Maximilians-Universitaet in Munich who was involved in the morphological analysis of the specimens, said in a statement.