Cover Story：Single molecule fluorescence resonance energy transfer (smFRET) is a technique used to study the conformational change of a molecule by detecting the transfer efficiency of fluorescence energy between donor and acceptor fluorescence inside a single molecule. However, to obtain the information of these biological macromolecules, the statistical analysis of a large number of single molecular signals is required. Manual analysis is time-consuming and laborious and lacks objectivity and reproducibility. Therefore, the wavelet transform and rolling ball algorithms are applied to smFRET images to analyze single molecule signals. Based on the accurate detection of single molecule signals, we analyzed the linearity of the images processed by the rolling ball and wavelet transform algorithms. The results show that the two methods can not only remove the background noise of smFRET images but also maintain the linearity of single molecule fluorescence signals. At the end of this paper, we draw a statistics histogram of 15 bp DNA FRET efficiency and calculated the FRET efficiency value using rolling ball algorithm processing.