Generally, biosensors are designed to translate physical, chemical, or biological events into measurable signals, thus offering qualitative and/or quantitative information regarding the target analytes. While the biosensor field has received considerable scientific interest, integrating this technology with microfluidics could further bring significant improvements in terms of sensitivity and specificity, resolution, automation, throughput, reproducibility, reliability, and accuracy. In this manner, biosensors-on-chip (BoC) could represent the bridging gap between diagnostics in central laboratories and diagnostics at the patient bedside, bringing substantial advancements in point-of-care (PoC) diagnostic applications. In this context, the aim of this manuscript is to provide an up-to-date overview of BoC system development and their most recent application towards the diagnosis of cancer, infectious diseases, and neurodegenerative disorders.