<p>Lignocellulosic biomass being one of the largest yet most underutilized renewable resources on the earth, is a major global concern. Physical and chemical pretreatment methods for valorizing these resources can be economically non-viable, energy-intensive and environmentally detrimental; thus, laccase pretreatment holds a promising biocatalytic approach for strategic agro-waste valorization. This research focuses on environmentally friendly and cost-effective methods of optimized laccase production and its effect on lignocellulosic agro-waste degradation by <i>Pleurotus ostreatus</i>, cultivated on locally sourced substrates. In this study, two different fermentation methods, submerged and solid-state fermentation, were compared and meticulously evaluated for six indigenously produced agricultural waste residues such as paddy straw, sugarcane bagasse, corn cob, coconut husk, groundnut shells and sawdust, which widely occur in the western zone of India. Key physiological parameters influencing laccase production were optimized to obtain enhanced enzymatic yield. SEM imaging validated the significant breakdown of substrate structure by degrading lignocellulosic residues, indicating successful enzymatic removal of lignin. Among various substrates tested, Paddy straw emerged highly effective, yielding 70 U mL-1 crude laccase and, after partial purification, 181.89 U mL-1 via solid-state fermentation. The partially purified enzyme was further validated by Fast Protein Liquid Chromatographic (FPLC) method, SDS PAGE, followed by zymography. Notably, paddy straw enabled 95.04 ± 0.13% lignin degradation and a marked reduction in phenolic content, underscoring its potential for sustainable laccase production and biomass valorization. This study demonstrates circular economy-oriented research that focuses on substrate-driven optimization for a region-specific agro-waste utilization through enzymatic biocatalysis, offering lignocellulosic valorization and biomass pretreatment.</p>