In the present study, Tween 80, a non-ionic surfactant, has been used for enhanced hydrogen production by crude glycerol bioconversion using co-culture of Enterobacter aerogenes and Clostridium butyricum. The purpose of introducing the surfactant was to decrease the crude glycerol viscosity, so that apparent solubility and bioavailability of glycerol could be improved at the expenses of pretreatment steps. Experiments were planned using central composite design (CCD); crude glycerol and Tween 80 concentrations were optimized whereas, hydrogen production, glycerol utilization and viscosity of the media were considered as responses. The response surface for quadratic model showed, Tween 80 concentration had significant effect (p < 0.05) on all the three responses. Using the optimized conditions at 17.5 g/L crude glycerol and 15 mg/L Tween 80, hydrogen production reached a maximum of 32.1 ± 0.03 mmol/L of medium. The increase in hydrogen production was around 1.25-fold in presence of Tween 80 in comparison to its absence with 25.56 ± 0.91 mmol/L production. Selected optimum conditions were also validated against absence of crude glycerol (4.69 ± 0.76), with pretreated crude glycerol (20.06 ± 0.51) and across mono-culture system (15.43 ± 0.79 to 22.14 ± 0.94). Introduction of Tween 80 to the fermentation medium improved the glycerol utilization rate, resulting in increased hydrogen production and eliminated pretreatment steps.