Recently, a high-quality gray Akoya pearl has been so popular in the market that many treated gray pearls appeared, resulting in a mess. Among them, the color of pearls changed by γ-ray irradiation treatment is the most difficult to identify. Therefore, distinguishing the origin of those gray pearls has become an urgent problem to solve. We chose a few white and light yellow Akoya pearls as samples to be treated by different doses of γ -ray irradiation. By observing the surface and drilled holes of these samples, the non-destructive spectroscopic test included photoluminescence spectra, UV-Vis spectra, and 3D fluorescence spectra to test and compare those unirradiated, irradiated, and naturally colored gray Akoya pearls. By comparing the amplification characteristics of samples, it is found that γ-ray irradiation samples have a light nacre and deep brown nucleus. In contrast, naturally colored ones show a brown interlayer of organic matter between a light nacre layer and white nucleus, and sometimes a “black spot” resulted from the concentrated area of organic matter. By comparing the spectroscopy characteristics of the samples, it is found that the fluorescence background of the photoluminescence spectra of the irradiated samples is higher than that of unirradiated and naturally colored ones. Their F/A value (1.34~1.98), the intensity ratio between the background peak and aragonite main peak, is slightly higher than that of naturally colored ones (0.52~1.12). The reflectance of UV-Vis spectra of irradiated samples is lower than unirradiated ones, absorbed at 360 nm in the UV region. In comparison, the absorption valley of the naturally colored samples located in the range of visible light at 430~530 nm, which changes with different overtone, sometimes there is a weak wide and weak absorption at 750~800 nm. 3D fluorescence spectra show that the luminescence center of irradiated and unirradiated samples is consistent, except fluorescence intensity reduces by half compared with unirradiated ones. However, the main luminescence center of the irradiated pearls is completely different from that of the naturally colored gray pearls. The strongest fluorescence center in 3D fluorescence spectra of γ-ray irradiated Akoya pearls is at Ex/Em of 374/449 nm and 463 min the form of double peaks, while that of the naturally colored gray Akoya pearl is at Ex/Em of 286 nm/340 nm. In a word, combined with the observation from the surface and the drill hole and 3D fluorescence spectra, it is well to distinguish between naturally colored gray Akoya pearls and γ -ray irradiated gray Akoya pearls. In contrast, the F/A value of photoluminescence spectra and UV-Vis spectra can be used as an auxiliary identification.