DNA analysis is still difficult to implement in high schools in Indonesia due to limitations of equipment and materials. Has conducted research that aims to analyze the CO1 gene in fruit flies, from pineapples and its application as a teaching material in DNA study in high school. CO1 gene amplification results pineapple fruit fly has a long 664 bp. The results of the analysis of alignment with the method of BLAST on NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) obtained pineapple fruit flies CO1 sequence have a level 99% similarity with Drosophila ananassae [JQ679117.1]. Reconstruction of phylogenies by Maximum Likelihood models indicate pineapple fruit fly has the closest kinship with Droshohila pseudoannanasae [AY757280]. Application as a teaching material in high school subject DNA produce learning outcomes and student learning activities in the very good category. Activities and student learning outcomes are also supported by the response of students and teachers to study media at the very good category.