Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented.