Aptamers are single-stranded nucleic acids that selectively bind to target molecules. Most aptamers are obtained through a combinatorial biology technique called SELEX. Since aptamers can be isolated to bind to almost any molecule of choice, can be readily modified at arbitrary positions and they possess predictable secondary structures, this platform technology shows great promise in biosensor development. Over the past two decades, more than one thousand papers have been published on aptamer-based biosensors. Given this progress, the application of aptamer technology in biomedical diagnosis is still in a quite preliminary stage. Most previous work involves only a few model aptamers to demonstrate the sensing concept with limited biomedical impact. This Critical Review aims to summarize progress that might enable practical applications of aptamers for biological samples. First, general sensing strategies based on the unique properties of aptamers are summarized. Each strategy can be coupled to various signaling methods. Among these, a few detection methods including fluorescence lifetime, flow cytometry, upconverting nanoparticles, nanoflare technology, magnetic resonance imaging, electronic aptamer-based sensors, and lateral flow devices have been discussed in more detail since they are more likely to work in a complex sample matrix. The current limitations of this field include the lack of high quality aptamers for clinically important targets. In addition, the aptamer technology has to be extensively tested in a clinical sample matrix to establish reliability and accuracy. Future directions are also speculated to overcome these challenges.