Fluorescence polarization/anisotropy (FP/FA) approaches are appealing for targets sensing in homogeneous solution due to simplicity, reproducibility and sensitivity. Taking advantage of aptamers, aptamer structure switch FA methods are unique for small molecule detection based on the competition between aptamer-target binding and the hybridization of aptamer and complementary DNA (cDNA). However, usually small FA change is generated in these aptamer assays that only rely on size change caused by hybridization of an oligonucleotide because of the rapid local rotation of fluorophores and small mass change. Here we describe a simple and general aptamer structure switch FA assay for small molecules by employing a large-sized streptavidin (SA) as an effective signal amplifier based on proximity effect to reduce local rotation of fluorophore. In this design, the SA-labeled cDNA hybridizes with fluorescein (FAM)-labeled aptamer, drawing FAM close to SA and bringing a much higher FA value due to restricted local rotation of FAM. Small molecule-aptamer probe binding causes displacement of the SA-labeled cDNA and great decrease of FA. The closeness of SA to FAM in the duplex is key for this proposed strategy to produce large FA changes in target detection. Our method enabled to detect 60 pM aflatoxin B1 (AFB1), 1 nM ochratoxin A (OTA), and 0.5 μM adenosine triphosphate (ATP), respectively. This aptamer FA method combines the merits of aptamers and FA analysis, and it is promising in applications of detection of small molecules with good sensitivity.