The goal of microphysiological systems (MPS) is to replicate the relevant functionality of human organ tissues in in vitro. MPS technology so far has been used to simulate the various human organs and with the help of sensor integration in the MPS systems the biological activities of the organ to be modeled have been translated into data to be analyzed for further considerations. Most standard characterization approaches are intrusive and detrimental, and not feasible for online monitoring of cell cultures. Microfluidic biosensors, for instant, provide non‐invasive on‐line detection of biomarkers and molecules under targeted indicators with a high detection extent, successfully overcoming the limits of existing approaches. Microfluidic biosensors are rapidly being incorporated into MPS and employed for real‐time target identification as a result. In this review the focus is on emerging ways for miniaturizing and embedding biosensing systems in MPS also known as “organ‐on‐chip”. Cutting‐edge microfluidic biosensors are also covered with examples, showing their key benefits in monitoring MPS and highlighting current breakthroughs, before describing the remaining problems and anticipated future improvements in integrated microfluidic biosensors.