Abstract: By analyzing the 168 experimental-confirmed σ54 promoter sequences, two conservative regions that are -24 and -12 regions are obtained. The trimer frequency at 17 positions in these conservative regions is selected as inputting parameter. By adding pseudo-count into position weight matrix, the σ54 promoter can be predicted. The 168 negative sequences are extracted from coding regions and convergent intergenic regions. In Jackknife cross-validation, the overall accuracy reaches to 82.0%, suggesting that the model can be further used in the theoretical and experimental study of σ54 promoter.