The objective of this study was to assess the feasibility of noninvasive alcohol testing in vivo with near-infrared (NIR) spectroscopy. The suitable distance between light source and detector was determined by Monte-Carlo simulation. The NIR spectra signals of alcohol in vitro and in vivo were measured, and the blood alcohol concentration (BAC) was measured with breath test method. Wavelet de-noising and partial least squares (PLS) method were used to establish the quantitative calibration model of alcohol. The results indicate that alcohol spectra had two absorption peaks at range of 2200nm~2400nm. The optimal principal component number of PLS model is 3, RMSEP=9.29, MREP=3.5%,R=0.9802. The model has good prediction accuracy. NIRS might provide a new method to the measurement of alcohol in vivo.