Organs-on-chips have emerged as viable platforms for drug screening and personalized medicine. While a wide variety of human organ-on-a-chip models have been developed, rarely have there been reports on the inclusion of sensors, which are critical in continually measuring the microenvironmental parameters and the dynamic responses of the microtissues to pharmaceutical compounds over extended periods of time. In addition, automation capacity is strongly desired for chronological monitoring. To overcome this major hurdle, in this protocol we detail the fabrication of electrochemical affinity-based biosensors and their integration with microfluidic chips to achieve in-line microelectrode functionalization, biomarker detection and sensor regeneration, allowing continual, in situ and noninvasive quantification of soluble biomarkers on organ-on-a-chip platforms. This platform is almost universal and can be applied to in-line detection of a majority of biomarkers, can be connected with existing organ-on-a-chip devices and can be multiplexed for simultaneous measurement of multiple biomarkers. Specifically, this protocol begins with fabrication of the electrochemically competent microelectrodes and the associated microfluidic devices (~3 d). The integration of electrochemical biosensors with the chips and their further combination with the rest of the platform takes ~3 h. The functionalization and regeneration of the microelectrodes are subsequently described, which require ~7 h in total. One cycle of sampling and detection of up to three biomarkers accounts for ~1 h.