SummaryIn this work, we introduce persistent homology for the analysis of cryo‐electron microscopy (cryo‐EM) density maps. We identify the topological fingerprint or topological signature of noise, which is widespread in cryo‐EM data. For low signal‐to‐noise ratio (SNR) volumetric data, intrinsic topological features of biomolecular structures are indistinguishable from noise. To remove noise, we employ geometric flows that are found to preserve the intrinsic topological fingerprints of cryo‐EM structures and diminish the topological signature of noise. In particular, persistent homology enables us to visualize the gradual separation of the topological fingerprints of cryo‐EM structures from those of noise during the denoising process, which gives rise to a practical procedure for prescribing a noise threshold to extract cryo‐EM structure information from noise contaminated data after certain iterations of the geometric flow equation. To further demonstrate the utility of persistent homology for cryo‐EM data analysis, we consider a microtubule intermediate structure Electron Microscopy Data (EMD 1129). Three helix models, an alpha‐tubulin monomer model, an alpha‐tubulin and beta‐tubulin model, and an alpha‐tubulin and beta‐tubulin dimer model, are constructed to fit the cryo‐EM data. The least square fitting leads to similarly high correlation coefficients, which indicates that structure determination via optimization is an ill‐posed inverse problem. However, these models have dramatically different topological fingerprints. Especially, linkages or connectivities that discriminate one model from another, play little role in the traditional density fitting or optimization but are very sensitive and crucial to topological fingerprints. The intrinsic topological features of the microtubule data are identified after topological denoising. By a comparison of the topological fingerprints of the original data and those of three models, we found that the third model is topologically favored. The present work offers persistent homology based new strategies for topological denoising and for resolving ill‐posed inverse problems. Copyright © 2015 John Wiley & Sons, Ltd.