Nymphaea hybrid is a large tropical water lily in the genus Nymphaea. Different from the common water lily at present, N. hybrid has a long flowering period, beautiful flower shape, rich flower color, pleasant fragrance and rich biological active substances, which has attracted more and more attention. The flower color of different N. hybrid varies with the change of flowering stage, but the change rule of flower color is not clear yet. In this study, the outer petals of four different colors of N. hybrid flowers at different flowering stages were used as the test materials. The chroma value, anthocyanin, flavonoids, pH value of cell fluid, contents of soluble sugar (SS) and soluble protein (SP), and activities of phenylalanineammonialyase (PAL) and chalconeisomerase (CHI) enzymes were measured during the flowering process. The epidermal cells of most petals of the N. hybrid flower showed irregular folds and protrusions. The pigment of purple, pink and yellow N. hybrid flowers was mainly distributed in the upper and lower epidermal cells, while the pigment distribution was almost absent in white N. hybrid flower. With the progress of flowering period, the total anthocyanin (TA) content, PAL and CHI activity decreased, cell pH value increased gradually. The contents of total flavonoids (TF), soluble sugar and soluble protein increased first and then decreased, while petals lightness L^* increased, and there were significant differences among parameters at different flowering stages. Total anthocyanin in petals was negatively correlated with L^* and pH of cell fluid, but positively correlated with PAL and CHI enzyme activities. This study shows that the changes of flower color of N. hybrid are affected by multiple factors, and the presentation of flower color is related to the shape of petals and the position of anthocyanin distribution. The content of anthocyanin and flavonoids in petals directly affect the depth of flower color. The activities of PAL and CHI enzymes promote the formation of flower color of N. hybrid to a certain extent. Meanwhile, pH, soluble sugar, and soluble protein affect the formation and stability of the flower color of N. hybrid by participating in physiological metabolism during the flowering process, and then affect the change of flower color tone of N. hybrid.