In order to establish and optimize the technology system of AFLP for Ethanoligenens harbinense’s genome,in this study several key factors influencing AFLP were studied and optimized in detail.The results showed that the effect was better when purity and concentration of template DNA(OD260/OD280) were 1.8-1.9 and 50-100 ng respectively in 25 μL reaction system.The best dosage and time of restriction enzyme digestion of(EcoRI/MseI) were EcoRI(2.0 μL)/MseI(0.7 μL) and 3 h respectively.Furthermore,agar gel electrophoresis map analysis also showed that the bands,which were amplified by 7 different primer combinations of EcoRI-A/MseI-G,EcoRI-G/MseI-A,EcoRI-G/MseI-C,EcoRI-T/MseI-A,EcoRI-T/MseI-C,EcoRI-T/MseI-G and EcoRI-T/MseI-T,were clear and stable without disturbed background and could be used to express the polymorphism distribution of DNA fingerprinting of Ethanoligenens harbinens clearly.