Drosophila melanogaster is one of the important test organisms in genetics thanks to its fast growth rate in a culture. This study demonstrates that the fly D. melanogaster can also be exploited as a source for nanofiber production in biotechnical applications. First, its chitin content was determined (7.85%) and then high molecular weight chitosan (141.4 kDa) was synthesized through deacetylation of chitin isolates. Chitosan nanofibers with the diameter of 40.0073 ± 12.347 nm were produced by electrospinning of Drosophila chitosan. The physicochemical properties of obtained chitin and chitosan from D. melanogaster were determined by Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FT-IR). The study demonstrated that the fly D. melanogaster can be utilized for production of chitosan nanofiber concerning its cultivability and low-cost culture requirements.