Abstract: Three-dimensional fluorescence spectroscopy is a multi-dimensional detection method in many fields such as environmental sciences and biology, especially in classifying organic matter types. It is often used in combination with other spectrometers to analyze the structural characteristics of samples. In this paper, the authors took three types of gray Akoya pearl with different cause of colour (naturally coloured, γ-ray irradiated, dyed)as the research objects to explore the application of three-dimensional fluorescence spectroscopy in the study of pearls. The results showed that these three types of gray Akoya pearls can be accurately identified by a three-dimensional fluorescence spectrum. These samples have different combinations of luminescence center distribution, and the highest intensity fluorescence center at λ_ex/ λ_em: 374 nm/448 nm and 462 nm are the warning evidence of the treated samples. According to the division of dissolved organic matter corresponding to the fluorescence center, all the samples have naturally coloured ammonia, protein, and humic acid. The test result of naturally coloured gray Akoya pearls found that the nacre layers of both gray and white Akoya pearls were composed of tryptophan and protein components, while the organic matter layer was mainly composed of humus. The nacre layer with organic matter shows a peak of humus, indicating that some organic matter has infiltrated the nacre. Humic acid is a complexing agent of many trace metal elements, which can promote the complexation of metal ions with proteins in pearls. The three-dimensional fluorescence spectrum of γ-ray irradiated gray Akoya pearls show that the main organic components are humus. Still, the overall fluorescence intensity of the irradiated sample is much weaker than that of naturally coloured gray Akoya pearls, which may be due to the degradation of organic components of humic acids caused by γ-ray irradiation. The three-dimensional fluorescence spectra of dyed gray Akoya pearl samples show aromatic protein, which were not showned in other types of samples, may caused by dye.