Aptamers are bioaffinity molecules with many advantages, including in vitro generation, low cost, and a wide acceptance of chemical modification. Combined with transduction technologies, aptamers can be further utilized to develop aptasensors, which have superior merits of low cost, rapid response, high sensitivity and selectivity, etc. Thus, aptamers and aptasensors have excellent bioapplication potential in the field of analytical chemistry, in areas such as disease diagnosis, food analysis, environment monitoring, etc. This Special Issue is devoted to the dissemination of research on aptamer generation and bioapplication. We will cover a wide range of advanced studies on aptamer-related exploration in the scope of this Special Issue.