Tracking and differentiating small insects at the individual levels requires appropriate marking materials because of their small size. This study proposes and investigates the use of fluorescent silica nanoparticles (FSNPs) as an internal marker owing to their good optical properties and biocompatibility. FSNPs were prepared using the water-in-oil reverse microemulsion technique with Rubpy dye as a fluorophore. The obtained particles were spherical, monodispersed in nanosize and exhibited bright orange luminescence under ultraviolet (UV) light. Internal marking was accomplished in fruit flies (Drosophila melanogaster) through feeding. The result shows that the fruit flies exhibit bright luminescence in their abdomen when exposed to UV light. The marking persistence duration of FSNPs in the fruit fly bodies is longer than those of other fluorescent dyes. Fruit flies fed with FSNPs have a longer lifespan than those fed with Rubpy dye. There was no difference in fertility and negative geotaxis response among the treatment and control groups. These findings demonstrate that FSNPs can be used as an internal marker in fruit flies, and are possibly applied with other small insects with a translucent abdomen.