A new method was developed for sensitive determination and quantitative analysis of acetoin in beer in our laboratory. Identification of derivative of acetoin (diacetyl) was carried out by headspace sampling-gas chromatography (HS-GC). The optimal equilibrium time of diacetyl was 32 min for the mixture of 4.5 mL iron reagent and 0.5 mL beer with the stirring of a shaker. As compared to 3 level calibrations, data obtained by 6 level calibrations were more accurate and consistent, and its correlation coefficient was between 0.99 and 0.999. The measuring precision for diacetyl was significantly improved to ⩽2% R.S.D (relative standard deviation) under the condition of HS (30 psi)/GC (22 psi) when compared to HS (30 psi)/GC (25 psi). Detecting precision of two level temperatures was ⩽1.5% R.S.D, which made all the detecting values existing between UCL (upper control limit) and LCL (lower control limit) in comparison with one level configuration. It was more reliable and suitable for the analysis of diacetyl to set up temperature configuration of two levels in HS-GC. Our results suggested that this method could be used successfully to analyse the concentration of acetoin in beer.