The preparation of self-assembled DNA nanostructure with different sizes and shapes has been one of the most promising research areas in recent years, while the application of these DNA nanostructures in biosensors is far from fully developed. Here, we presented a novel carrier system to construct an electrochemiluminescence (ECL) aptasensor for ultrasensitive determination of lipopolysaccharides (LPS) on the basis of self-assembled tetrahedron DNA dendrimers. Doxorubicin (Dox), a well-known intercalator of double stranded DNA (dsDNA), was conjugated with the ECL luminophore of N-(aminobutyl)-N-(ethylisoluminol) (ABEI) to form a new type of ECL indicators (Dox-ABEI), which could noncovalently attach to dsDNA through intercalation. Based on this property, self-assembled tetrahedron DNA dendrimers were employed as an efficient nanocarrier to achieve a high loading efficiency for Dox-ABEI with significantly amplified ECL signal output. Streptavidin (SA) and biotin, a typical ligand–receptor pair, has been chosen to anchor the tetrahedron DNA dendrimers on the electrode surface. Moreover, by converting LPS content into DNA output, catalyzed hairpin assembly (CHA) target recycling signal amplification strategy was also adopted to enhance the sensitivity of the ECL aptasensor. With combining the loading power of the tetrahedron DNA dendrimers for ECL indicators, the inherent high sensitivity of ECL technique and target recycling for signal amplification, the proposed strategy showed a detection limit of 0.18 fg/mL for LPS.