A MOF-based bio-bar code material was synthesized and firstly applied to develop an electrochemical streptomycin (STR) aptasensor. By using MOF-based bio-bar code and enzyme-assisted target recycling for dual-signal amplification, highly sensitive detection of STR was achieved. The sensing surface was simply fabricated by immobilizing a mixed monolayer of thiolated cDNA/aptamer duplexes (dsDNA) and 6-mercapto-1-hexanol (MCH) on the gold nanoparticle modified screen printed carbon electrode (Au/SPCE). The presence of target STR caused highly efficient removal of the aptamers from dsDNA assisted by Exo I enzyme. Then MOF-based bio-bar codes were backfilled to achieve the adsorption of electroactive Ru(NH3)63+ (RuHex) on electrode surface. The electrochemical signal of the surface-confined RuHex was used for quantitation. The analytical performance for STR was satisfactory with a wide linear range of 0.005–150 ng mL−1, a low detection limit of 2.6 pg mL−1 and a good selectivity towards other three antibiotics. Moreover, the application of this aptasensor for determination of STR in real milk samples was also realized. With these merits, this dual-signal amplification assay might provide one of the effective ways for food safety monitoring. Display omitted •A MOF-based bio-bar code material was synthesized and firstly applied to develop a sensitive streptomycin aptasensor.•Different signal amplification strategies were integrated in one sensing protocol to push down the detection limit.•This aptasensor was successfully applied in detecting streptomycin in real milk samples.