An electrochemical tetrahedral APT biosensor (Au-TAN biosensor) was successfully constructed, which had a stable structure and excellent performance using tetrahedral DNA nanomaterials (TDN) and Au nanoparticles (AuNPs) for the detection of acetamiprid. Specifically, we designed a partial paired complementary DNA (cDNA) of acetamiprid aptamer and inserted the cDNA together with the acetamiprid aptamer into four single-stranded DNA sequences forming each side of the TDN, modifying AuNPs to increase the current signal intensity, to assemble the Au-TAN. This proposed aptasensor was compared with ssAPT aptasensor and tetrahedral APT nanostructures (TAN) aptasensor, respectively. The results showed that the performance of the designed aptasensor was better than those of the above two aptasensors. The detection limit of the aptasensor was calculated to be 0.077 pM (S/N = 3), which was far lower than that of other methods that we compared. Under optimized conditions, the proposed aptasensor showed a wide linear detection range from 0.1 pM to 10 nM. The Au-TAN biosensor opens up new ideas for the construction of aptasensors and provides a new method for detection of small molecular substances, such as pesticides residues.