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Phytoplankton assemblages are essential for understanding the quality of primary production in marine ecosystems. Here, we describe the development of a methodology for monitoring marine phytoplankton assemblages using an in situ multi-wavelength excitation fluorometer (MEX). The MEX recorded the fluorescence excited with nine light-emitting diodes, temperature, and sensor depth. We prepared reference datasets comprising MEX fluorescence and plant pigment-based phytoplankton assemblages of nine chemotaxonomy groups (diatoms, dinoflagellates, cryptophytes, chlorophytes, haptophytes type 3, haptophytes type 4, prasinophytes, cyanophytes, and prochlorophytes). Conversions from the MEX fluorescence to the phytoplankton assemblages were conducted with two processes. First, target MEX fluorescence was decomposed using a linear inverse model for calculating coefficients. Second, pigment-based chemotaxonomy of the target MEX fluorescence was reconstructed using the coefficients and the chemotaxonomy assemblages of the reference data. Cross-validation analyses indicated good estimation of the proportion of diatoms, dinoflagellates, cryptophytes, cyanophytes, and prochlorophytes with MEX, and when chlorophytes, haptophytes and prasinophytes were summarized as other eukaryotes, the positive correlation was seen between proportions estimated with MEX and pigments as same as other five chemotaxonomy groups. Repeated MEX observations were conducted in the Kuroshio, the Sea of Japan, the Oyashio, and the Okhotsk Sea. The water-column integrated biomass indicated that the diatoms were an important primary producer in the Oyashio and the Okhotsk Sea, while eukaryotes were important in the Sea of Japan and prochlorophytes were important in the Kuroshio. Our method with the MEX will be a powerful tool to understand and estimate the chemotaxonomy-level assemblages and biomass in the ocean.