The aim of this study was to remove 5-hydroxymethyl furfural (5-HMF) and furfural, known as fermentation inhibitors, in acid pretreated hydrolysates (APH) obtained from Scenedesmus obliquus using activated carbon. Microwave-assisted pretreatment was used to produce APH containing glucose, xylose, and fermentation inhibitors (5-HMF, furfural). The response surface methodology was applied to optimize key detoxification variables such as temperature (16.5–58.5 °C), time (0.5–5.5 h), and solid–liquid (S-L) ratio of activated carbon (0.6–7.4 w/v%). Three variables showed significant effects on the removal of fermentation inhibitors. The optimum detoxification conditions with the maximum removal of fermentation inhibitors and the minimum loss of sugars (glucose and xylose) were as follows: temperature of 36.6 °C, extraction time of 3.86 h, and S-L ratio of 3.3 w/v%. Under these conditions, removal of 5-HMF, furfural, and sugars were 71.6, 83.1, and 2.44%, respectively, which agreed closely with the predicted values. When the APH and detoxified APH were used for ethanol fermentation by S. cerevisiae, the ethanol produced was 38.5% and 84.5% of the theoretical yields, respectively, which confirmed that detoxification using activated carbon was effective in removing fermentation inhibitors and increasing fermentation yield without significant removal of fermentable sugars.