The focus of present study is to explore cotton stalk as potential source for bioethanol production. In this context, cotton stalk was subjected to series of treatment includes dual stage acid hydrolysis, followed by detoxification and fermentation subsequently. Detoxified hydrolysate obtained after first two treatment, having a sugar concentration of 11 g/L corresponds to yield of 0.396 g/g of biomass was exposed to fermentation by co-culture of Saccharomyces cerevisiae MTCC 36 and Pachysolen tannophilus MTCC 1077. Parametric optimization study reveals that test organisms in association utilized 93.84 % of total available sugars in semi aerobic mode of fermentation having pH 5.5, inoculated with 10 % (v/v) co culture inoculum, incubated with 120 rpm for first 24 h and then kept in static mode at 30 oC till 48 h, produced maximum ethanol concentration of 4.96 g/L with fermentation efficiency of 87.52 %. The obtained yield was recorded as 0.446 g/g of available sugar for fermentation (0.179 g/g of biomass).