This study aimed to evaluate the possibility of cotton waste enrichment with glycine betaine (GB) for production of two strains (P9, P10) of king oyster (Pleurotus eryngii). Cotton waste was used as (100%) Control, (T = Cotton waste) and augmented with various combinations of GB, (T = 2 mM/L, T = 4 mM/L, T = 6 mM/L, T = 8 mM/L and T = 10 mM/L). The response of king oyster to GB was evaluated by earliness, yield, biological efficiency (BE), minerals (N, P, K, Zn, Cu, Mg, Mn, Fe, Na, Ca), Total sugars, TSS, Reducing sugars, non-reducing sugars, ascorbic acid, proximate (crude protein, carbohydrates, crude fibers, ash, fats) content of fruiting body and Fourier Transform Infrared spectroscopy (FTIR) analysis compared with the control substrate (Cotton waste).The earliness, yield, and BE were higher as compared to control substrate and increased with an augmentation in the concentration of GB within the cotton waste. Two strains showed (on dry weight basis) 33.9-54.9 mg/g N, 6.8-12.5 mg/g P, 16.9-25.1 mg/g K, 40.5-64.2 mg/kg Zn, 17.1-37.3 mg/kg Cu, 1174-1325 mg/kg Mg, 20.1-29.1 mg/kg Mn, 129-265 mg/kg Fe, 779-835 mg/kg Ca), 6.3-11.3% total sugars, 7.3-14.9 Brix TSS, 2.1-7.3% reducing sugars, 10.4-18.1% crude protein, 3.6-4.4% crude fiber and 5.6-16.7 mg/100g on various concentration of GB enrich cotton waste. Cotton waste enriched with GB significantly affected nutritional profile of king oyster mushroom.The results revealed that GB enriched cotton waste can be used as an innovative substrate to enhance the yield and quality of king oyster mushroom. This article is protected by copyright. All rights reserved.