In this study, a new surface-enhanced Raman scattering (SERS)-based method has been developed for the detection of plasmin activity. Firstly, different peptide sequences, which are specific to plasmin, were examined. Then, SERS substrates were prepared by chosen peptide substrate. Enzyme activity was determined by pursuing the reduction of DTNB band at 1331 cm with Raman spectroscopy. The reduction in SERS intensity was related to the plasmin activity, and changes in SERS intensity vs. plasmin concentration graph was obtained. Limit of detection (LOD) and limit of quantification (LOQ) values were calculated as 2.14 U/mL and 6.42 U/mL, respectively. Intra- and inter-day repeatability results were determined as 1.45% and 1.47% relative standard deviation (RSD). Also, recovery of the method was determined for the plasmin spiked milk samples. The results demonstrated that the proposed method could be successfully used to detect the plasmin activity in milk samples.