In this study, a new quantification method was developed for analysis of ethanol in whole blood samples by needle extraction (NeedlEx^®) and gas chromatography/mass spectrometry (GC/MS). Whole blood samples (0.1 mL) containing ethanol and ethanol-d₆ (internal standard) were incubated at room temperature in a 10-mL headspace vial. The NeedlEx^® for alcohols with a gas aspirating pump was exposed to the headspace of vial to allow adsorption alcohols before GC/MS. The GC separation of the compounds was achieved on a fused-silica capillary column Rtx-5 ms (30 m × 0.25 mm i.d.; 0.25-μm film thickness) with MS detection operated in electron impact ionization ion source mode. The regression equations showed good linearity (r>0.998) from 0.1 to 5.0 mg/mL for whole blood. The accuracies and precisions were 99.0–112% and 3.0–7.0%, respectively. The method was successfully applied to actual analyses of autopsy samples. The present results on the analysis of ethanol by NeedlEx^®-GC/MS suggest its applicability to a number of other volatile compounds in clinical and forensic toxicology.