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In the last decades, the use of electrochemical and electrode modification-based biosensors has aroused great research interest in widely different applications such as medical diagnostics, drug analysis, environmental monitoring, food analysis, and biochemical analysis. Moreover, the use of various nanomaterials for electrode modifications and the production of these modifications based on the layer-by-layer film deposition techniques have provided significant advantages in the production of stable and sensitive sensors that provide a large surface area. In this review, the applications of electrochemical methods developed for the determination of major biomarkers that are important in the diagnosis of different diseases are discussed. In three main categories, diseases and related biomarkers, electrochemical sensors and modification strategies, and applications of prepared electrodes according to the layer-by-layer strategy are described. Current biosensors, operating conditions, some validation characteristics, and applications reported to detect several biomarkers in the last four years are presented to the researchers in a table. Moreover, conclusions and future perspectives are presented, highlighting some ongoing challenges and some topics for improvement to maximize sensor sensitivity.