In the present study, the green Biocellulose Nanofibers (BC), a vitally emerging biomaterial, was produced by fermentation of wheat straw (WS), as a widely available agricultural waste, using. Two different fermentation methods were used; Separate Hydrolysis and Fermentation (SHF), and Simultaneous Saccharification and Fermentation (SSF). Different acidic and enzymatic WS pretreatment conditions were used to understand the effect of pretreatment conditions on BC production. Afterward, sugar hydrolsates were simultaneously or separately inoculated with Gluconacetobacter Xylinum bacterium (i.e., for SSF and SHF, respectively), at optimum production conditions in shake flasks for 7 days to produce the biocellulose nanofibers. BC productions of 9.7 g/L in SHF and 10.8 g/L in SSF were achieved when WS was pretreated with dilute acids. Enzymatic treatment of WS after acidic pretreatment increased sugars’ concentrations from the hydrolysis, which increased BC production in SHF to 10.6 g/L. However, enzymes in SSF broke cellulose I alpha linkage in BC and decreased its production compared to no enzymatic treatment. Results show that glucose extracted from WS (~55% of total sugars) was found essential for the cellular metabolism, while xylose (~28% of total sugars) was highly consumed during cells growth phase. Generally, increasing thermal treatment, time and temperature, resulted in increasing furfural concentration. This observed to inhibits bacterial cells growth and leads to lower nanofibers yield when exists at concentration higher than 1 g/L threshold.In general, results obtained in the present study demonstrate the ability of utilizing agricultural wastes in the fermentation production of BC. Such a step is expected to eliminate cost of expensive pure sugars as a carbon source in the fermentation. Also the study shows an improved production yield by using effective fermentation techniques as SSF compared to classical methods used in literature.