Abstract The main concern of lignocellulosic biomass utilization for biofuel production is the presence of lignin which hinder the hemicellulose and cellulose accessibility. In this study, chemical and biological pretreatments have been used for decomposition of the lignocellulosic cotton stalk (CS) into monosaccharides. Long-term fermentation/co-fermentation (upto 5 cycles) of pretreated CS by immobilized yeasts (Saccharomyces cerevisiae YPH499 and Pachysolen tannophilus 32691) for bioethanol was investigated. Spectroscopic analysis (including FTIR, XRD, SEM, and TGA) showed the disintegration and abrasion in CS structure after application of both the pretreatments. The maximum sugar utilization efficiency in 1st cycle of co-fermentation by immobilized yeasts was 94.1 and 90.4% with 0.46 and 0.44 g/g bioethanol production in chemical and biological pretreatment, respectively. Moreover, bioethanol yield was slightly sustained till 2nd cycle (0.38−0.40 g/g). However, bioethanol production steadily declined at 3rd cycle and reached to the lowest value at 5th cycle. These results demonstrated that co-fermentation with immobilization approach might significantly improve the bioethanol production from pretreated lignocellulosic biomass (including CS).