The effect of binding between the lignin isolates from an alkali (NaOH)− and an acid (H2SO4)− pretreated Miscanthus and cellulolytic enzymes in Cellic® CTec2 was investigated. Additonally, cellobiose and Avicel were enzymatically hydrolyzed with and without lignin isolates to study how enzyme binding onto lignin affects its conversion to glucose. Three carbohydrate−lignin loadings (0.5:0.25, 0.5:0.5, and 0.5:1.0% (w/v)) were employed. The results indicated that β-glucosidase (BG) had a strong tendency to bind to all lignin isolates. The overall tendency of enzyme binding onto lignin isolate was similar regardless of pretreatment chemical concentration. Though enzyme binding onto lignin isolates was observed, hydrolysis in the presence of these isolates did not have a significant (p > 0.05) impact on glucose production from cellobiose and Avicel. Cellobiose to glucose conversion of 99% was achieved via hydrolysis at both 5 and 10 FPU/g carbohydrate. Hydrolysis of Avicel with 5 and 10 FPU/g CTec2 resulted in 29.3 and 47.7% conversion to glucose, respectively.