Lipopolysaccharides (LPS), integral components of the outer membrane of all gram-negative bacteria, are closely associated with foodborne diseases such as fever, diarrhea and hypotension and thus, the early and sensitive detection of LPS is necessary. In this study, an aptasensor assay based on hybridization chain reaction (HCR) was developed to detect LPS. Briefly, two complementary stable species of biotinylated DNA hairpins coexisted in solution until the introduction of a detection probe triggered a hybridization chain reaction cascade. The DNA conjugates specifically reacted with the LPS, which were captured by the ethanolamine aptamer attached to the reaction well surface. After optimizing the key reaction conditions, such as the reaction time of HCR, the amount of the capture probe and detection probes, the increase in the LPS concentration was readily measured by the optical density value and a relatively low detection limit (1.73 ng/mL) was obtained, with a linear response range of 1–10⁵ng/mL. The approach presented herein introduced the use of an aptasensor for LPS discrimination and HCR for signal amplification, offering a promising option for detecting LPS.