Bioconversion of CO/syngas to produce ethanol is a novel route in bioethanol production, which can be accomplished by some acetogens. Specific culture vessels and techniques are needed to cultivate these microorganisms since they are anaerobic and substrates are gaseous. In this work, gas-sampling bag was applied as a gas-cultivation system to study CO/syngas bioconversion by Clostridium autoethanogenum and was demonstrated to be efficient because of its flexibility and excellent ability to maintain the headspace atmosphere. C. autoethanogenum can use CO as the sole carbon and energy source to produce ethanol, acetate as well as CO2. In the experimental range, higher ethanol production was favored by higher yeast extract concentrations, and the maximum ethanol concentration of 3.45g/L was obtained at 1.0g/L of yeast extract. Study with various bottled gases showed that C. autoethanogenum preferred to use CO other than CO2 and produced the highest level of ethanol with 100% CO as the substrate. C. autoethanogenum can also utilize biomass-generated syngas (36.2% CO, 23.0% H2, 15.4% CO2, 11.3% N2), but the process proceeded slowly and insufficiently due to the presence of O2 and C2H2. In our study, C. autoethanogenum showed a better performance in the bioconversion of CO to ethanol than Clostridium ljungdahlii, a strain which has been most studied, and for both strains, ethanol production was promoted by supplementing 0.5g/L of acetate.