The demand for cheap, healthy, and sustainable alternative protein sources has turned research interest into microbial proteins. Mycoproteins prevail due to their quite balanced amino acid profile, low carbon footprint and high sustainability potential. The goal of this research was to investigate the capability of Pleurotus ostreatus to metabolize the main sugars of agro-industrial side streams, such as aspen wood chips hydrolysate, to produce high-value protein with low cost. Our results indicate that P. ostreatus LGAM 1123 could be cultivated both in a C-6 (glucose)- and C-5(xylose)-sugar-containing medium for mycoprotein production. A mixture of glucose and xylose was found to be ideal for biomass production with high protein content and rich amino acid profile. P. ostreatus LGAM 1123 cultivation in a 4 L stirred-tank bioreactor using aspen hydrolysate was achieved with 25.0 ± 3.4 g L−1 biomass production, 1.8 ± 0.4 d−1 specific growth rate and a protein yield of 54.5 ± 0.5% (g/100 g sugars). PCA analysis of the amino acids revealed a strong correlation between the amino acid composition of the protein produced and the ratios of glucose and xylose in the culture medium. The production of high-nutrient mycoprotein by submerged fermentation of the edible fungus P. ostreatus using agro-industrial hydrolysates is a promising bioprocess in the food and feed industry. © 2023 by the authors.