Biodemulsifier is a new type of demulsifiers for breaking oil-water emulsion. One demulsifier-producing strain, Alcaligenes sp. S-XJ-1 could grow on waste diesel oil (WDO), dry weight of the strain was up to 2.0 g/L after being cultivated for 7 d, 10 g/L S-XJ-1 cell suspension reduced surface tension of water from 72.0 mN/m to 29.7 mN/m. Biodemulsifier produced by S-XJ-1 was 0.3 g/L, its critical micelle concentration (CMC) was 150 mg/L, showing a better surface activity than chemical surfactant SDS. Furthermore, it showed an demulsifying efficiency over 70% for W/O model emulsion. It was indicated S-XJ-1 could utilize C14-C20 n-alkanes composing waste diesel oil by GC-MS, C20 n-alkane was almost completely comsumed by S-XJ-1 with utilization ratio of 99%. In addition, n-alkanes utilization ratio and demulsifying capability increased when length of carbon chain increased. Both utilization ratio and demulsifying capability of biodemulsifier produced by S-XJ-1 using C20 n-alkane as the carbon source were superior to other n-alkanes, and similar to waste diesel oil. It was identified the biodemulsifier produced by S-XJ-1 using waste diesel oil was lipopeptide by TLC and FTIR.