Phytoplankton are extraordinarily diverse, comprising 13 phylogenetic groups, with diatoms, dinoflagellates, and haptophytes among the most prominent eukaryotes in the ocean. Development of sensor technologies for rapid taxonomic and physiological analysis of phytoplankton communities is crucial for ecological monitoring programs in the global ocean. We describe a novel, ultra-sensitive, multi-spectral fluorescence induction and relaxation instrument (a mini-FIRe) and examine its analytical capability of rapidly determining phytoplankton taxonomic groups, as well as physiological characteristics and photosynthetic rates. We collected and analyzed the database of spectral and photosynthetic properties of major taxonomic groups of phytoplankton. We revealed that the spectral shape of the functional absorption cross-section of Photosystem II (PSII), sPSII(lex), is remarkably constrained within each major phylogenetic group of eukaryotic phytoplankton, including diatoms, haptophytes, dinoflagellates, and chlorophytes. Variability in sPSII(lex) within each group was significantly smaller than the difference between groups. We also examined the classical excitation spectra of chl a fluorescence yields, Fm(lex). Our comparative analysis revealed that sPSII(lex) is a better and more specific proxy for taxonomic analysis. For instance, our developed sPSII-based algorithm correctly identified 90% of experimental data, compared to 77% identified by the Fm-based algorithm. Our results suggest that the multi-color variable fluorescence analysis offers a tool for combined physiological and taxonomic analysis, including identification of major phyla within the ‘red’ lineage of eukaryotic phytoplankton.