•Lateral flow nucleic acid biosensor was constructed for rapid detection of microRNAs.•Three target microRNAs were all successfully detected with good linearity and sensitivity.•MicroRNAs were detected in the spiked serum samples with this lateral flow nucleic acid biosensor. The authors describe a rapid and low-cost approach for multiplex microRNA(miRNA) assay on lateral flow nucleic acid biosensor (LFNAB). The principle of assay is based on sandwich-type nucleic acid hybridization reactions to produce gold nanoparticle (GNP)-attached complexes (ssDNA-microRNA-ssDNA/GNPs), which are captured and visualized on the test zone of LFNAB. By designing three different test zones on LFNAB, simultaneous detection of microRNA-21, microRNA-155 and microRNA-210 was achieved with an adding-measuring model by using GNP as visual tag. The method was challenged by testing the microRNAs in spiked serum samples with satisfied results. In our perception, the test is a particularly valuable tool for clinical application and biomedical diagnosis, particularly in limited resource settings.