In recent years, the need for simple, fast, and economical detection of food and environmental contaminants, and the necessity to monitor biomarkers of different diseases have considerably accelerated the development of biosensor technology. However, designing biosensors capable of simultaneous determination of two or more analytes in a single measurement, for example on a single working electrode in single solution, is still a great challenge. On the other hand, such analysis offers many advantages compared to single analyte tests, such as cost per test, labor, throughput, and convenience. Because of the high sensitivity and scalability of the electrochemical detection systems on the one hand and the specificity of aptamers on the other, the electrochemical aptasensors are considered to be highly effective devices for simultaneous detection of multiple-target analytes. In this review, we describe and evaluate multi-label approaches based on (1) metal quantum dots and metal ions, (2) redox labels, and (3) enzyme labels. We focus on recently developed strategies for multiplex sensing using electrochemical aptasensors. Furthermore, we emphasize the use of different nanomaterials in the construction of these aptasensors. Based on examples from the existing literature, we highlight recent applications of multiplexed detection platforms in clinical diagnostics, food control, and environmental monitoring. Finally, we discuss the advantages and disadvantages of the aptasensors developed so far, and debate possible challenges and prospects.