A sensitive, and single step reaction assay of glycoproteins in antibody-free mode was proposed on a 4-mercapto-phenylboronic acid (4-MPBA) modified silver nanoparticle film (AgNF) by using surface enhanced Raman scattering (SERS). 4-MPBA/AgNF was designed both as a glycoprotein-specific recognition biosensor and as a Raman reporter for the SERS platform. Variation in the ratio I1574/I1586 for 4-MPBA/AgNF in response to glycoprotein capture constitutes the SERS assay which exhibits high sensitivity and selectivity against nonglycoproteins and is shown to function in complex samples.