Freshwater nucleated pearls have gained a significant share in the pearl market, due to their large size, good roundness, rich color, and strong luster. However, there is still no explicit conclusion on color-causing mechanism in colored freshwater nucleated pearls, which is attributed to the fact that there are very few studies currently focused on the mechanism research. Herein, we filtrate multiple colors of freshwater cultured pearls to study the mechanism via laser Raman spectroscopy and colorimetry. The Raman spectroscopy revealed these samples possess three different C—C and CC peaks, exhibiting interesting corresponding relationship, which means the concurrence of C—C (1 123～1 125 cm-1) corresponding to the CC (1 506～1 509 cm-1), the C—C (1 132～1 134 cm-1) corresponding to the CC (1 522～1 523 cm-1) or the CC (1 525 cm-1), the C—C (1 128～1 131 cm-1) corresponding to the CC double peak (1 506～1 509 and 1 520～1 523 or 1 525 cm-1). Owing to the differences of CC peaks in the Raman spectra, the samples were classified into P, T, G, P+T, T+G, and P+G. Combining with the chromatic parameters, we explored the relationship between the lightness (L) or saturation (S) and the CO3/C—C peak area ratio in the same type, displaying the positive correlation between the lightness (L) and the CO3/C—C peak area ratio for P-type pearls. In contrast, the negative correlation was found between the saturation (S) and the CO3/C—C peak area ratio for P, T, and G types, which confirmed that the three different C—C and CC peaks were caused by different color-causing substances. Moreover, we attributed color-causing organic compounds to linear polyenes by Raman spectroscopy. The Raman peaks of linear polyenes show redshift with the increase of orderliness. Linear polyene molecules with peaks closer to the infrared region have more extended and straighter carbon skeletons, which leads to the difference between the peaks of C—C and CC in the Raman spectra of different samples. We also categorised the colored freshwater nucleated pearls via chromatic parameters. The pearls with hue angle (h)∈(0, 15), (15, 45), (335, 360) are classified as the red series, yellow series, and purple series, respectively.