Gold nanoparticles (AuNPs) labeled lateral-flow test strip immunoassay (LFTS) has been widely used in biomedical, feed/food, and environmental analysis fields. Conventional ILFS assay usually uses spherical AuNPs as labeled probes and shows low detection sensitivity, which further limits its widespread practical application. Unlike spherical AuNP used as labeled probe in conventional ILFS, in our present study, a hierarchical flowerlike AuNP specific probe was designed for LFTS and further used to detect Escherichia coli O157:H7 (E. coli O157:H7). Three types of hierarchical flowerlike AuNPs, such as tipped flowerlike, popcornlike, and large-sized flowerlike AuNPs were synthesized in a one-step method. Compared with other two kinds of Au particles, tipped flowerlike AuNPs probes for LFTS particularly exhibited highly sensitive detection of E. coli O157:H7. The remarkable improvement of detection sensitivity of tipped flowerlike AuNPs probes can be achieved even as low as 10(3) colony-forming units (CFU)/mL by taking advantages of its appropriate size and hierarchical structures, which is superior over the detection performance of conventional LFTS. Using this novel tipped flower AuNPs probes, quantitative detection of E. coli O157:H7 can be obtained partially in a wide concentration range with good repeatability. This hierarchical tipped flower-shaped AuNPs probe for LFTS is promising for the practical applications in widespread analysis fields.