National Basic Research Program of China [2010CB732402, 2013CB933703]; National Science Foundation of China [91313302, 21205100, 21275122, 21075104]; National Instrumentation Program [2011YQ03012412]; Natural Science Foundation of Fujian Province, China [2013J01061]; Fundamental Research Funds for the Central Universities [2012121025]; National Science Foundation for Distinguished Young Scholars of China [21325522] ; A novel method, monoclonal surface display SELEX (MSD-SELEX), has been designed for simple, rapid, efficient, and cost-effective enrichment and identification of aptamers from a library of monoclonal DNA-displaying beads produced via highly parallel single-molecule emulsion PCR. The approach was successfully applied for the identification of high-affinity aptamers that bind specifically to different types of targets, including cancer biomarker protein EpCAM and small toxin molecule aflatoxin B-1. Compared to the conventional sequencing-chemical synthesis-screening work flow, MSD-SELEX avoids large-scale DNA sequencing, expensive and time-consuming DNA synthesis, and labor-intensive screening of large populations of candidates, thus offering a new approach for simple, rapid, efficient, and cost-effective aptamer identification for a wide variety of applications.