Aptamers are three-dimensional structures of DNA or RNA that present high affinity and selectivity to specific targets, obtained through in vitro screening. Aptamers are used as biological recognizers in electrochemical biosensors, the so-called aptasensors, providing greater specificity in recognizing the most diverse analytes. Electrochemical aptasensors have extremely relevant characteristics, such as high sensitivity, low cost compared to other biorecognizers such as antibodies, and excellent compatibility, being considered one of the most promising alternative methods in several areas, such as biomedical diagnosis and monitoring environmental contaminants. In this sense, the present work reviews the relevant aspects of methodologies based on electrochemical aptasensors and their applications in determining antibiotics, seeking to foster innovation in electrochemical biosensors.