In this study, we investigated carbon isotopic fractionation during production of ethanol and acetic acid from carbon sources in anaerobic batch type of bioreactor using C. ljungdahlii which was known as a main ethanol production microbial under anaerobic condition. We hypothesized that isotopic fractionation (δ¹³C values) could vary in anaerobic fermentation by Clostridium. ljungdahlii. We adopted carbon sources (fructose, synthesis gas, ¹³CO gas) to compare ethanol and acetate production under various condition included substrate concentration, gas composition, pH, and partial pressure. Results showed that initial pH varied from 7.25 to 4.45 as anaerobic bacteria producing ethanol and acetic acid. Gas chromatograph data showed that CO and H2 concentration decreased as CO2 production was increased with ethanol and acetate production. We also found that ethanol and acetate concentration were promoted by adding fructose, high pressure of H2 and CO in the culture media. The maximum concentration of ethanol and acetate were 0.72 and 1.6g/L. When injecting fructose and gaseous type of substrate with different conditions, ranges of δ13C values of microbe showed from -21.86∼-19.27‰ (initial values) to -19.31∼-17.8‰ (final values). These results can trace where these carbon resources came from and carbon pathways in anaerobic fermentation with isotopic fractionation effect.