A biosensor is a sensing device that incorporates a biological sensing element and a transducer to produce electrochemical, optical, mass, or other signals in proportion to quantitative information about the analytes in the given samples. The microfluidic chip is an attractive miniaturized platform with valuable advantages, e.g., low cost analysis requiring low reagent consumption, reduced sample volume, and shortened processing time. Combination of biosensors and microfluidic chips enhances analytical capability so as to widen the scope of possible applications. This review provides an overview of recent research activities in the field of biosensors integrated on microfluidic chips, focusing on the working principles, characteristics, and applicability of the biosensors. Theoretical background and applications in chemical, biological, and clinical analysis are summarized and discussed.