Global interest in lignocellulosic biorefineries has increased in the recent past due to technological advancements in sustainable and cost-effective production of numerous commodity and speciality chemicals and fuels from renewable lignocellulosic biomass (LCB). As a result, the market value of biorefinery products has also increased over the time, with an estimated worth of USD 867.7 billion by 2025. However, biorefinery operations, especially enzymatic hydrolysis, suffer from many challenges that limits the cost-effectiveness of conversion of LCB. Therefore, it is essential to understand and address these challenges in future biorefineries. The paper focuses on recent trends and challenges in enzymatic hydrolysis of LCB during lignocellulosic biorefinery operation for greener synthesis of energy, fuels, chemicals and other high-value products. Insights into the gaps in knowledge and technological challenges have also been addressed together with focus on future research needs and perspectives of enzymatic hydrolysis of LCB for biorefinery applications.