Abstract Alkaline pretreatment causes the disruptions in the lignin structure and breaks the linkage between lignin and the other carbohydrate fractions in lignocellulosic biomass. The aim of the current study was to investigate the possibility of rye straw hydrolysis with 2% aqueous ammonia for 0–8 h, at 60 °C and 90 °C, under atmospheric pressure. The study investigated how rye straw hydrolysis with aqueous ammonia at low concentration and relatively low temperatures affect such parameters as total and volatile solids (TS & VS), chemical oxygen demand (COD) and pH at low concentration. The experimental results indicated that the concentration of reducing sugars and volatile fatty acids (VFA) in rye straw after 8 h hydrolysis with ammonia were about 1.3 and 5.2 times higher at 60 °C, respectively and 1.8 and 5.2 times higher at 90 °C, respectively compared to samples in which water hydrolysis of rye straw. The percentage content of glucose in the hydrolysates ranged between 17.98% and 50.98% in water and 21.62%–41.75% in 2% aqueous ammonia. SEM and FTIR analysis of rye straw after 8 h hydrolysis confirmed the positive effect of 2% aqueous ammonia on changes in the material structure.