Cheese whey powder (CWP) is an attractive raw material for ethanol production since it is a dried and concentrated form of CW and contains lactose in addition to nitrogen, phosphate and other essential nutrients. In the present work, deproteinized CWP was utilized as fermentation medium for ethanol production by Kluyveromyces fragilis. The individual and combined effects of initial lactose concentration (50–150 kg m−3), temperature (25–35 °C) and inoculum concentration (1–3 kg m−3) were investigated through a 23 full-factorial central composite design, and the optimal conditions for maximizing the ethanol production were determined. According to the statistical analysis, in the studied range of values, only the initial lactose concentration had a significant effect on ethanol production, resulting in higher product formation as the initial substrate concentration was increased. Assays with initial lactose concentration varying from 150 to 250 kg m−3 were thus performed and revealed that the use of 200 kg m−3 initial lactose concentration, inoculum concentration of 1 kg m−3 and temperature of 35 °C were the best conditions for maximizing the ethanol production from CWP solution. Under these conditions, 80.95 kg m−3 of ethanol was obtained after 44 h of fermentation.