Tetracycline (TC) is widely used to treat bacterial infections in humans and animals due to its low price and good antibacterial properties. The abuse of tetracycline has led to TC residues in daily food that could seriously affect human health. Thus, it is imperative to develop highly sensitive and selective methods for TC detection. In this work, we developed a colorimetric method for TC detection based on an enzyme-linked aptamer assay (ELAA) with multivalent HRP-mimicking DNAzyme. An aptamer was used as an alternative recognition element in the enzyme-linked immunosorbent assay (ELISA). Multivalent HRP-mimicking DNAzyme, assembled via hybridization chain reactions (HCR), was used for catalytic substrate color rendering in ELAA. The multivalent HRP-mimicking DNAzyme exhibited enhanced catalytic capacity and improved the detection sensitivity greatly. The limit of detection was 8.1 × 10-2 ng mL-1 with a linear range from 1.0 × 10-2 ng mL-1 to 1.0 × 104 ng mL-1 toward TC in buffer. To challenge the practical application capability of this strategy, the detection of TC in milk samples was also investigated and showed similar linear relationships. Due to the introduction of an aptamer, this ELAA strategy shows high selectivity towards TC and has potential for the detection of a wide spectrum of analytes.