With super properties, such as photoluminescence properties, edge effect, low cytotoxicity and great biocompatibility, graphene quantum dots (GQDs) have attracted great attention in biological and biomedical applications. The potential toxicity investigations of GQDs still need to involve. Few studies have been illuminated that GQDs could alter the function and structure of trypsin. The molecular interaction between trypsin and GQDs was systematically researched through the combination of multi-spectroscopic and chemometrics approaches. The fluorescence quenching experiment showed that GQDs quench the intrinsic fluorescence of trypsin and inhibit the biological activity of trypsin. When different concentrations of GQDs were added, the fluorescence emission peak intensity of trypsin at 350 nm continuously decreased and had a blue shifted (350 to 344 nm), indicating that GQDs could change the microenvironment of trypsin and increase its hydrophobic. Meanwhile, the higher the concentration of GQDs, the more obvious the change of trypsin fluorescence, indicating that GQDs interacts and changes the secondary structure of the macromolecule. The microenvironment of protein amino acid residues is determined by the conformation of protein molecules. The spiral structure of proteinase decreased from 19.12% to 16.23% in the circular dichroism experiment indicated that the addition of GQDs induced the alteration of the secondary structure of trypsin and relaxed the trypsin framework. The three-dimensional fluorescence further indicated that the conformation of trypsin changed with the addition of GQDs. When the microenvironment of the chromophores of serum albumin changes, its UV-visible absorption spectrum also changes. Due to the complex life system, most of the information in the test of spectrum data is implicit and overlapping. We need to use and develop the effective biological signal collection, transduction, data processing and analysis method to get the useful information that can explain life as much as possible from the measured data. To obtain sufficient and effective chemical information of life, this study adopts the continuous titration technique collecting multidimensional spectrum data. An expanded UV-Vis spectral data matrix was analyzed by the multivariate curve resolution-alternating least squares (MCR-ALS) chemometrics approach. To further understand the state and the whole dynamic change process of each component when GQDs and trypsin reached equilibrium in action, the qualitative (spectrum of each component) and quantitative (the changing trend of concentration) information were obtained from the heavily overlapped spectra. The analytical results of MCR-ALS provide a basis for further understanding of the kinetic process of the interaction between GQDs and trypsin, indicating that GQDs can interact with trypsin and form GQDs₁₅-trypsin complex. The results offered insights into the binding mechanism of GQDs with trypsin and significant information for possible toxicity risk of GQDs to human health.