The olive fruit fly, Bactrocera oleae, is a highly significant pest in olive growing countries, and controlling it may be enhanced by using genetically modified strains, especially for sterile insect technique programs. To improve and expand this technology, piggyBac-mediated germline transformation was achieved in a laboratory-adapted wild olive fruit fly strain. A piggyBac vector was used that is marked with both green (EGFP) and red (DsRed) fluorescent protein genes, with a duplicate piggyBac 5' terminal inverted repeat sequence inserted between the marker genes for subsequent immobilization of vectors integrated into the host genome. Five transformant G1 adults were selected based on marker gene expression, yielding an estimated minimum germline transformation frequency of approximately 1.8% per G0 adult. All transgenic lines expressed DsRed and EGFP, although DsRed was more visible and robust compared to EGFP expression, which remained stable for more than 20 generations. Marker expression and PCR analysis, including an insertion site sequence, was consistent with stable genomic insertions. This is the first study of B. oleae transformant lines to assess life fitness parameters, including egg hatching, larval survival, larval-to-pupal survival, pupal-to-adult survival, and fertility. In three transgenic lines, survival at all biological stages was similar; overall fitness was significantly lower compared to wild-type olive flies, but similar to fitness levels previously reported for transgenic Mexican fruit flies. The studies presented here demonstrate the development of marked strains for olive fly using polyubiquitin-regulated fluorescent proteins in transformation vectors that can be stabilized for strain stability and ecological safety. This is the first successful effort to establish transgenic strains for an important agricultural pest in Turkey.