Bio-cellulose is the microbial extracellular cellulose that is produced by growing several microorganisms on agriculture by-products, and it is used in several food applications. This study aims to utilize sago by-product, coconut water, and the standard medium Hestrin-Schramm as the carbon sources in the culture medium for bio-cellulose production. The bacteria Beijerinkia fluminensis WAUPM53 and Gluconacetobacter xylinus 0416 were selected based on their bio-cellulose production activity. The structure was determined by Fourier transform infrared spectroscopy and scanning electron microscopy, while the toxicity safety was evaluated by brine shrimp lethality test. The results of Fourier transform infrared spectroscopy showed that the bio-cellulose produced by B. fluminensis cultivated in sago by-products was of high quality. The bio-cellulose production by B. fluminensis in the sago by-product medium was slightly higher than that in the coconut water medium and was comparable with the production in the Hestrin-Schramm medium. Brine shrimp lethality test confirmed that the bio-cellulose produced by B. fluminensis in the sago by-product medium has no toxicity, which is safe for applications in the food industry. This is the first study to determine the high potential of sago by-product to be used as a new carbon source for the bio-cellulose production.