The advancement in early diagnosis and precise treatments options result in more predictable and powerful health care modalities. Aptamers are known as nucleic acid structures with three-dimensional conformation to selectively bind a target site. Physicochemical properties of aptamers, their conjugation with nanoparticles (NPs) in theranostics applications and their internalization have been found to be of interest in development of aptamer-based drug delivery systems. Therefore, we aimed to present an overview on the structure and generation of aptamers followed by advantages of aptamers-conjugated NPs and their theranostics applications in various diseases such as oncology, inflammatory diseases and viral diseases. Afterward, we discussed several reports on the internalization approaches of aptamers, efficiency of aptamers vs. their analogous, and implications of aptamers in clinical trials. Finally, we discussed the current challenges and future perspectives of actively targeted aptamers for clinical application. In conclusion, this review may hold a great promise for development of aptamer-based therapeutic platforms in clinical trials.