For decades, the sandwich assay has been a mainstay in the fields of biodetection, clinical diagnostics, environmental monitoring, and quality control in various industries. By eliminating the requirement of labeling the molecular target, the sandwich assays usually require the simultaneous binding of the recognition probe and signaling probe, which makes them extremely specific. Likewise, because signaling is typically coupled to an enzyme catalytic or amplified signaling mechanism, the sandwich assay usually achieves impressive sensitivity of detection. Along with progress in chemistry, biotechnology, and nanotechnology, the sandwich assay has been extensively developed. For example, the sandwich assay has been employed successfully for the detection of a spectrum of targets, including pathogens, proteins, nucleic acids, small molecules, and ions. The signaling mechanism has also been extensively expanded from radiolabeling to more readily detectable readouts such as enzyme catalysis, fluorescence, or electricity. Furthermore, the basic sandwich architecture has been adapted into a supersandwich platform, which usually further amplifies the signal and pushes the detection limit down. To date, thousands of papers have been published on the sandwich assay. However, to our best knowledge, no such review paper summarizing the developments and future goals of this field yet exists. Thus, we present a critical review of the literature on the sandwich assay in the past 10 years to summarize and comment upon its development and advances, concentrating mainly on recent developments in nanobiotechnology.