Today, many researchers are focusing on research for alternative promising energy sources and sustainable technology for bioethanol production to meet the increasing global energy demand. Here, we develop a novel one-pot pretreatment technology by co-immobilizing laccase, cellulase and beta-glucosidase to act as a tri-enzyme biocatalyst for evaluating the bioethanol production potential of four sustainable lignocellulosic biomasses viz., Typha angustifolia, Arundo donax, Saccharum arundinaceum, and Ipomoea carnea. The co-immobilized enzyme system was more stable at different temperatures and at longer storage, compared to free enzyme. During enzymatic saccharification, Saccharum arundinaceum showed higher total reducing sugar of 205 +/- 3.73 mg/g when compared to other biomass. The highest percentage of bioethanol yield of 63.43 +/- 9.35% was obtained with Ipomoea carnea. The effects of co-immobilized tri-enzyme biocatalyst on the biomasses were evaluated. The results revealed that the co-immobilized tri-enzyme biocatalyst could act as effective one-pot pretreatment for the production of bioethanol from lignocellulosic biomass.