In this design, we introduced a surface plasmon resonance (SPR) fiber-sensing probe into a column chromatography (CC) system to realize on-line dynamic detection in sample separation. The refractive index of the gel around the probe would be adjusted dynamically by the concentration change of the sample during CC separation. To demonstrate the separation and on-line detection process, bovine serum albumin (BSA) and riboflavin-5-phosphate sodium (FMN-Na) are chosen as the analytes in a Sephadex gel filtration chromatography system. The results show that the apparent reversible shift of the SPR spectrum can characterize the separation process. Specifically, the separated BSA with an outflow time of 8 min can cause a resonance wavelength shift of 15.5 nm, and the FMN-Na with an outflow time of 26 min can cause a shift of 8.4 nm. This on-line dynamic detection of SPR spectra has great potential to save time and simplify the analysis process compared to the complex thin layer chromatography detection steps in traditional manual CC.