The determination of the polyanionic drug heparin has been reported in the literature by using various spectral techniques at the expense of the polycationic drug protamine. But, the determination of protamine in the presence of heparin by optical methods has not been reported so far. Since the administration of protamine is associated with different side effects its accurate determination is essential in the presence of heparin. The present study reports the sensitive and selective determination of protamine in the presence of heparin by colorimetric, spectrophotometric and spectrofluorimetric methods using GQD capped silver nanoparticles (Ag-GQDs) for the first time. The yellow colour of the Ag-GQD containing heparin immediately turns red while adding 100 ng mL-1 protamine. The obtained color change is due to an increase in the Ag-GQD size from 9 to 15 nm which was confirmed by HR-TEM studies. On the other hand, the surface plasmon resonance (SPR) band of Ag-GQDs at 411 nm decreased and a new peak appeared at 520 nm after the addition of protamine. While adding each 10 ng mL-1 protamine to Ag-GQDs, the emission intensity at 451 nm increases linearly with a correlation coefficient of 0.9910 and the limit of detection was found to be 0.05 × 10-9 g mL-1 (S/N = 3). Furthermore, the selective determination of protamine was achieved in the presence of heparin and other common interferents using Ag-GQDs. The practical application of the present sensor was displayed by determining protamine in blood serum.