Comparative genomics has identified a large number of non-coding segments that have been highly conserved over hundreds of millions of years of vertebrate evolution. To examine their functions in vivo, we generated over 300 Drosophila transgenic lines, each containing a unique human conserved noncoding segment inserted upstream of a core promoter fused to a GAL4 or a QF gene, and then assessed their enhancer activities. We observed reporter GFP expression in one or more tissues of embryos and larvae in all segments tested. Therefore, it is evident that human conserved segments can function as enhancer in Drosophila. Indeed, our assay system is more powerful and effective than the previously described mouse assay, which detected reporter gene expression only in a half of tested segments. What is more, induced GFP expression is biased toward the brain and central nervous system of Drosophila. This result may imply that human conserved segments share some unknown enhancer functions despite lacking sequence homology.