In this work, a filtration cell for synchronized UV-vis spectroscopic and electrochemical impedance investigation of the membrane fouling process has been designed. The setup achieves in situ monitoring of the interfacial region during filtration by fiber-optic UV-vis spectroscopy and electrochemical impedance spectroscopy. The influence of ionic strength on protein fouling of membrane was investigated in detail. Results indicate that the flux decline rate decreased slightly when the ionic strengths increased from 0 to 10 and 100 mM during initial filtration stage, however, membrane fouling at higher ionic strengths became more serious at longer fouling duration. This was mainly due to the generation of concentration polarization (CP) and electroosmotic back flow (EOBF) for longer duration, which caused elevated concentration of salt ions near the membrane surface and additional filtration resistance, and accompanied by salting-out dehydration of protein on membrane surface. This is the process that occurs in protein fouling during long-term run, the hydration forces experience from enhancement to disappearance. This study not only expends our knowledge of the effect of ionic strength on protein fouling behavior but also provides a method for the in situ and real time characterization of membrane-solution interface and filtration performance.