In this report, a water soluble perylene diimide derivative (PDI-Glu) was designed and prepared for rapid and visual detection of protamine. The sensing performance and mechanism of this probe toward protamine was investigated systematically by absorption and emission spectroscopic techniques. The fluorescence quenching caused by the aggregation of PDI-Glu in the presence of protamine could be outputted by spectrometer and observed by naked eyes. This method showed an excellent selectivity towards protamine in comparison of other analytes. It also featured good water solubility, large operable range, fast and visualized detection. The detection limit of protamine was calculated to be 0.09 μg mL−1. Finally, this probe can be also applied in serum samples and monitoring trypsin activity.