Because of the similar chemical structure of various fluorophore groups and cell culture medium, the spectra heavily overlap, making it difficult to conduct the qualitative and quantitative analysis of single components directly. To begin, we use Rhodamine B and a calibration standard baffle fluorescence spectrophotometer to solve the problem. Moreover, the water Raman unit (RU350) is used for fluorescence intensity normalization, which increases the dynamic detection range of the instrument, and then the parallel factor analysis algorithm, which inherits the advantage of "second order" or "mathematical separation, " decomposes the entire dataset into four fluorescent components (TRY, NADH, FAD, and VB6), without the complex "chemical separation." The average recoveries of these fluorophores are in the range of (99.5%±6.5%)-(100.5%±6.4%), and the detection limits are in the range of 0.003-0.013 μg/mL, indicating that the method reaches the detection level of trace analysis. This method can meet the demand for the quantitative detection of metabolic fluorescent components in a culture medium, indicating a significant advantage in cell metabolism and immune research, medical diagnosis in vitro, and cell sorting.