The growing number of various diseases and the increase of environmental contamination are the causes for the development of novel methods for their detection. The possibility of the application of affinity-based biosensors for such purposes seems particularly promising as they provide high selectivity and low detection limits. Recently, the usage of hybrid antibody-aptamer sandwich constructs was shown to be more advantageous in terms of working parameters in comparison to aptamer-based and immune-based biosensors. This review is focused on the usage of hybrid antibody-aptamer receptor layers for the determination of clinically and environmentally important target molecules. In this work, antibodies and aptamer molecules are characterized and the methods of their immobilization as well as analytical signal generation are shown. This is followed by the critical presentation of examples of hybrid sandwich biosensors that have been elaborated in the past 12 years.