A simple fluorescent probe HBI-GR based on the combination of the fluorophore (p-HBI) in green fluorescent protein (GFP) and Guanine riboside (GR) for HSA was successfully synthesized. HBI-GR showed an obvious fluorescence enhancement toward HSA without interference from other proteins, amino acids, anions and commonly existing metal ions. HBI-GR exhibited high sensitivity towards HSA with a good linear relationship between the fluorescence intensity of HBI-GR and HSA concentration from 0 to 0.06mgmL-1. The limit of detection, based on a signal-to-noise ratio of 3, was 15.09ngmL-1, which was much lower than that of most other reported probes. HBI-GR was almost non-fluorescent because of the bond twisting in the exited state of chromophore HBI. After binding to the hydrophobic pocket of HSA, it showed an obvious fluorescence enhancement due to the rigidifying of the flexible chromophore HBI by the hydrophobic environment. The resulting HBI-GR/HSA system also showed a satisfactory sensing ability toward trypsin through decreased fluorescence intensity with the detection limit of 0.0282ngmL-1. The fluorescence decreasing process was occurred as the lysine and arginine amino acids residues of HSA were cleaved by trypsin, which led to further exposure of HBI-GR to the PBS buffer phase and a concomitant decrease of the HBI-GR fluorescence intensity. Moreover, the probe HBI-GR was successfully used to detect HSA in healthy human urine and human blood serum samples. The practical application of the HBI-GR/HSA system for trypsin detection in healthy human urine also achieved satisfactory result.