Membrane fouling is the major drawback of membrane-based technologies because it will lead to severe flux declines and the need to clean or replace the fouled membrane. A technique capable of early diagnosis, process monitoring, and evaluation of the role of different foulants playing in the fouling process is crucial for the fouling control. We develop surface-enhanced Raman spectroscopy (SERS) as a new and versatile tool to investigate the fouling process of protein on PVDF (polyvinylidene fluoride) membranes as well as the fouling propensity of three different proteins. We optimized the aggregation level and volume of SERS-active Ag sol and the spectra acquisition method combined with a statistical analysis method to ensure a high detection sensitivity, signal uniformity, and stability. We then used SERS for the early diagnosis of the fouling process and determining when the membrane pores would be blocked. The fouled area was visualized by a combination of the silver staining and Raman mapping. The fouling propensity of different proteins was studied by comparing the relative SERS band intensities of different proteins on a glass slide and after membrane filtration. Compared with fluorescence-based techniques, the narrow, well-resolved Raman band, especially the use of the same excitation line and laser power, endows SERS the ability to compare the fouling propensity in a very simple way.