Background: The determination of drugs in pharmaceutical formulations and human biologic fluids is important for pharmaceutical and medical sciences. Successful analysis requires low sensitivity, high selectivity and minimum interference effects. Current analytical methods can detect drugs at very low levels but these methods require long sample preparation steps, extraction prior to analysis, highly trained technical staff and high-cost instruments. Biosensors offer several advantages such as short analysis time, high sensitivity, real-time analysis, low-cost instruments, and short pretreatment steps over traditional techniques. Biosensors allow quantification not only of the active component in pharmaceutical formulations, but also the degradation products and metabolites in biological fluids. The present review gives comprehensive information on the application of biosensors for drug discovery and analysis. Moreover, this review focuses on the fabrication of these biosensors.