Ganoderma lucidum triterpenoids (GLT) have shown special anti-tumor effects, but due to low yields and their hydrophobic structure, they have not found much clinical application. Box Behnken Design (BBD) developed a formulation to optimize the effective parameters in encapsulating GLT. Then, Gelatin nanofibers were characterized by SEM, 1H-NMR, and FTIR. Finally, the GLT release kinetics and GLT nanofibers cytotoxicity was studied. BBD shows the best values obtained are the solvent ratio of 11.5%, gelatin concentration of 22%, and voltage of 20 kV which were validated by an experimental assay. The results showed that the positively charged ionic groups present on the surface of gelatin adsorbed the carboxyl groups in GLT and the magnetic fields created by their nucleus influenced each other. Finally, GLT nanofibers with an average size distribution of 75.4 nm were observed. The result showed an efficiency of 75% for drug entrapment. The release kinetics demonstrated a sustained release of GLT follows the Korsmeyer-Peppas model that suggests a combination of surface drug dissolution and quasi-Fickian diffusion. Also, GLT nanofibers showed a higher cytotoxic activity against MCF-7 cell lines than free GLT. The generated model suggests a new approach to prediction and experimental nanofibers.