In vivo fluorescence methods are efficient tools for studying the distribution of phytoplankton in nature. Different algae species usually have different pigments with different ratios, which results in different fluorescence emission spectra. Based on multiple excitation wavelength fluorescence emission spectra, a discrimination technique is established in this study. The discrimination method, established by multivariate linear regression and weighted least-squares, was used to differentiate the samples cultured in the laboratory and collected from Jiaozhou Bay near Qingdao at the division level. The correctly discriminated samples were ≥ 86% for single algae samples, ≥ 88% for simulatively mixed ones, ≥ 91% for physically mixed ones and 100% for samples collected from Jiaozhou Bay. The result in this research is more definite for the physically mixed samples in the laboratory. The method described here can be employed to monitor the phytoplankton population in the marine environment.