A novel disposable three electrodes blood alcohol biosensor strip was fabricated by a screen printing technique. Multi-wall carbon nanotube(MWCNT), Meldola′s(MB), alcohol dehydrogenase(ADH)and nicotinamide adenine dinucleotide cofactor (NAD+) were modified on the surface of the carbon working electrode. Then hydrophilic membrane was stuck in the outermost of the three electrodes to make a reaction camera of 5 μL. Experimental results indicated that the biosensor possessed good accuracy and stability, the linear response range was 0.5-20 mmol/L with correlation coefficient of 0.9949, detection limit was 0.22 mmol/L, and the response time was less than 15 s. Some influencing factors to the biosensor were investigated, such as the pH, temperature and interferences. Correlation analysis showed that there was a significant correlation between the methods of biosensor and the headspace vapor phase chromatography in 10 whole blood samples(r=0.97583). Small volume whole blood sucked using siphonage to detect blood alcohol directly and quantitatively was the obvious character of the biosensor.