Objective To solve an unusual death case and develop a method for determining concentration of acetone in blood or urine samples from the dead body by headspace gas chromatography (HS-GC), which is quite helpful under certain circumstance when autopsy can’t be performed because of social customs, religious belief or other reasons. Methods A 67-year-old man, who had diabetes, was found dead in a hotel room and his family didn’t agree to perform an autopsy. In order to determine the cause of death, the blood and urine samples were sent to laboratory for poison and drugs analysis, but no common poison was found. Based on the excellent volatility, acetone in blood or urine samples was analyzed by a head-space gas chromatography system with hydrogen fire ionization detector (HS-GC/FID). Qualitative analysis result was obtained according to retention time, and quantitative analysis was performed by internal standard (tert-butyl alcohol) method. The average peak area derived from three replicate injections of each sample was used for the calculation of acetone concentration in blood or urine. Results Concentration of acetone in blood and urine was 6.89 mg/100mL and 57.1 mg/100mL respectively, both were much higher than the normal value. Conclusions Determination of acetone in blood or urine by HS-GC/FID provides a useful evidence for determining the manner of death, which is especially effective when short of pathology data. In such casework, thorough information about the dead should be taken into consideration to ensure the scientific deduction.