Lignin is the most abundant aromatic natural polymer and comprises about 25% of straw biomass. Nanolignin biosynthetic production method is a simple method and safely better than chemical or physical methods. It has an interest in using lignin in more advanced applications. In particular, lignin-based nanoparticles could find potential application use in functional surface coatings, nano glue, drug delivery, microfluidic devices, and food additive. This study aimed to optimize Lactobacillus bulgaricus in nano lignin synthesis, the effect of the incubation period and freeze-drying on the quality of the nanolignin. Lignin particles were biosynthesized using rice straw and Lactobacillus bulgaricus in a dark place with a temperature of 37°C for 24 hours, 48 hours, and 72 hours. Lignin nanoparticle was characterized using Fourier Transformer Infrared Spectroscopy (FTIR), Particle Size Analyzer (PSA), Scanning Electron Microscope (SEM) and Energy Dispersive X-ray (EDX). Theresults indicated that nano lignin has a spherical and amorphous shape. The average size of particles is 101.6 nm with an incubation period of 24 hours, 57.2 nm with an incubation period of 48 hours, and 276.9 nm with an incubation period of 72 hours. The incubation periods affect the size and shape of nanolignin and also show that the lignin chemical structure is within the nanoparticle formation process. Samples using freeze-drying enable have natural antibacterial compounds and have phenolic fragments containing recommended for nano preservatives.