Abstract: The widespread implementation of fifth-generation (5 G) technology has raised concerns regarding its impact on biosafety. This study aims to uncover metabolic alterations in Drosophila melanogaster in response to electromagnetic radiation through metabolomics under 3.5 GHz radiofrequency electromagnetic fields (RF-EMF) conditions. Newborn male flies were continuously exposed to 3.5 GHz RF-EMF at intensities of 0.1 W/m2, 1 W/ m2, and 10 W/m2 throughout their entire life cycle under 24-hour constant darkness. Metabolomic analysis was performed on Drosophila samples after long-term exposure. The results indicate that long-term exposure to RFEMF leads to disruptions in four metabolic pathways - alanine, aspartate and glutamate metabolism, tryptophan metabolism, ascorbate and aldehyde metabolism, and purine metabolism - as well as 34 differential metabolites across various power density in Drosophila melanogaster. Notably, the most significant metabolites (GABA, glucose-6-phosphate, AMP, N-formylglycinamide nucleotide, LPC, and MG) exhibited high sensitivity to RF-EMF showing significant decreases. The suggests that long-term exposure to electromagnetic wave treatment may reduce the overall metabolic levels in fruit fly. These findings provide valuable insights into the metabolic disturbance induced by RF-EMF and enhance our understanding of the potential health effects associated with 5 G radiation.