Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on cooperative binding split aptamers (CBSAs). CBSAs contain two target-binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two binding domains, enabling EATR signal amplification through exonuclease III's apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine. We further developed an instrument-free colorimetric assay employing EATR-mediated aggregation of CBSA-modified gold nanoparticles for the visual detection of low-micromolar concentrations of cocaine. On the basis of the generalizability of CBSA engineering and the robust performance of EATR in complex samples, we believe that such assays should prove valuable for detecting small-molecule targets in diverse fields.