Poor antimatrix interference capability of aptamers is one of the major obstacles preventing their wide applications for real-sample detections. Here, we devise a multiple-function interface, denoted as a nanoscale affinity double layer (NADL), to overcome this bottleneck via in situ simultaneous target enrichment, purification, and detection. The NADL consists of an upper aptamer layer for target purification and sensing and a lower nanoscale solid-phase microextraction (SPME) layer for sample enrichment. The targets flowing through the NADL-functionalized surface are instantly million-fold enriched and purified by the sequential extraction of aptamer and SPME. The formation of the aptamer-target complex is greatly enhanced, enabling ultrasensitive detection of targets with minimized interference from the matrix. Taking the fiber-optic evanescent wave sensor as an example, we demonstrated the feasibility and generality of the NADL. The unprecedented detection of limits of 800, 4.8, 40, and 0.14 fM were, respectively, achieved for three representative small-molecule targets with distinct hydrophobicity (kanamycin A, sulfadimethoxine, and di-(2-ethylhexyl) phthalate) and protein target (human serum albumin), corresponding to 2500 to 3 × 108-fold improvement compared to the sensors without the NADL. Our sensors also showed exceptionally high target specificity (>1000) and tunable dynamic ranges simply by manipulating the SPME layer. With these features comes the ability to directly detect targets in diluted environmental, food, and biological samples at concentrations all well below the tolerance limits.