Simultaneous detection of multiple microRNAs (miRNAs) with high sensitivity can give accurate and reliable information for clinical applications. By uniformly anchoring hairpin probes on the surface of DNA nanolantern, a three-dimensional DNA nanostructure contains abundant and adjustable modification sites, highly integrated DNA nanoprobes were designed and developed as catalytic hairpin assembly (CHA)-based signal amplifiers for enzyme-free signal amplification detection of target miRNAs. The nanolantern-based CHA (NLC) amplifiers, which were facilely prepared via a simple “one-pot” annealing method, showed enhanced biostability, improved cell internalization efficiency, accelerated CHA reaction kinetics, and increased signal amplification capability compared to the single-stranded DNA hairpin probes used in traditional CHA reaction. By co-assembling multiple hairpin probes on a DNA nanolantern surface, as-prepared NLC amplifiers were demonstrated to work well for highly sensitive and specific imaging, expression level fluctuation analysis of two miRNAs in living cells, and miRNAs-guided tumor imaging in living mice. The proposed DNA nanolantern-based nanoamplifier strategy might provide a feasible way to promote the cellular and in vivo applications of nucleic acid probes. A DNA nanolantern-based catalytic hairpin assembly (CHA) nanoamplifier strategy is developed for simultaneous detection of multiple microRNAs in living cells and in living mice. Display omitted •A DNA nanolantern-based catalytic hairpin assembly nanoamplifier strategy is developed.•The nanoamplifiers are easily prepared and show many advantages compared to traditional nucleic acid probes.•The nanoamplifiers achieve the sensitive imaging and expression level fluctuation analysis of two miRNAs in living cells.•The nanoamplifiers work well for miRNAs-guided tumor imaging in living mice.•The proposed strategy might promote the cellular and in vivo applications of nucleic acid probes.