Gabriel pearls are freshwater-nucleated pearls developed in recent years cultured in the mantle of Hyriopsis cumingii. In response to its coloration mechanism, scholars have proposed diversified viewpoints. In our research, both laser Raman spectroscopic and micro-infrared spectroscopic analyses were performed on Gabriel pearls with carotenoid standard samples (β-carotene and astaxanthin) as reference materials for a better understanding of the coloration mechanism, which was expected to provide a scientific theoretical basis for quality evaluation and cultivation technology of these pearls. The results confirmed the strong 1509 and 1126 cm ^-1 Raman peaks of the nacre of freshwater colored nucleated pearls attributed to ν₁(C=C) and ν₂(C—C) stretching vibration modes of carotenoid-binding protein, respectively. The presence of a weak peak at 1017 cm ^-1 was associated with the plane oscillation of CH₃ (ν₃). Raman spectra in the range of 2100--3800 cm ^-1 revealed five groups of peaks with varying intensities, which were attributed to frequency multiplication and combined frequency vibration of ν₁, ν₂, and ν₃. Notably, there was a correlation between the number of conjugated double bonds of carotenoid molecules or carotenoid-binding proteins and the color of freshwater-nucleated pearls, indicating one of the reasons for the coloration diversity of pearl color.