Cancer stem-like cells are closely related with the development and metastasis of tumors. Herein, an electrochemical method is proposed to identify stem-like cells in breast tumor. The core concept of the method is the use of multifunctional nanofibers (MNFs), which are synthesized through facile self-assembly of peptide probes. MNFs can perform three functions, specifically targeting surface biomarker to identify stem-like cells, recruiting silver nanoparticles (AgNPs) to generate electrochemical signals, and providing large amounts of reaction sites to amplify signals. Specially, breast cancer stem cells (BCSCs) are first captured by nucleolin aptamer immobilized on the electrode surface and then selectively recognized by MNFs through the binding with CD44, thereby offering a large number of azide groups for signal labeling. By tracing electrochemical signals from MNF-recruited AgNPs, the method demonstrates to detect target cells as low as 6 cells/mL within a wide linear range from 10 to 5 × 105 cells/mL. Moreover, the method can not only recognize BCSCs with high selectivity in complex environment but also monitor drug-induced stemness changes with high sensitivity, providing promising prospective clinic applications in the future.