Bacterial nanocellulose (BNC) can be produced using a variety of substrates as fermentation medium for use in various biomaterial applications. This study aimed to optimize the production of and characterize the BNC derived from lemongrass leaves (L-BNC) obtained by symbiotic culture of bacteria and yeast (SCOBY) kombucha. The lemongrass leaves (10, 15, and 20 g/L) and sugar (30, 50, and 70 g/L) were incubated for 14 d at 30 °C. The optimal treatment was used to ferment kombucha for 21 days at 30 °C, with initial SCOBY inoculum of 3% w/v and kombucha of 10% v/v for the resulting L-BNC. The L-BNC was characterized using scanning electron microscopy-energy dispersive X-ray (SEM-EDX) spectroscopy, Fourier-transform Infrared spectroscopy (FT-IR), and X-ray diffraction analysis (XRD). The optimal parameters of the lemongrass kombucha fermentation production process were lemongrass content 10 g/L and sugar content 30 g/L with the incubation period of two weeks for 56.8 g/L of SCOBY production. The SEM analysis of L-BNC revealed a three-dimensional fibrous extremely fine network of randomly arranged nanofibrils with diameter of 163 ± 34 nm and hydrogen bonds present in L-BNC fibril units. Meanwhile, XRD results showed a crystallinity of 67.2%.