An in vivo three-dimensional fluorescence method for the determination of algae community structure was developed by parallel factor analysis (PARAFAC) and CHEMTAX. The PARAFAC model was applied to fluo-rescence excitation-emission matrix (EEM) of 60 algae species belonging to five divisions and 11 fluorescent components were identified according to the residual sum of squares and specificity of the composition profiles of fluorescent. By the 11 fluorescent components, the algae species at different growth stages were classified correctly at the division level using Bayesian discriminant analysis (BDA). Then the reference fluo-rescent component ratio matrix was constructed for CHEMTAX, and the EEM–PARAFAC–CHEMTAX method was developed to differentiate algae taxonomic groups. The correct discrimination ratios (CDRs) when the fluorometric method was used for single-species samples were 100% at the division level, except for Bacil-lariophyta with a CDR of 95.6%. The CDRs for the mixtures were above 94.0% for the dominant algae species and above 87.0% for the subdominant algae species. However, the CDRs of the subdominant algae species were too low to be unreliable when the relative abundance estimated was less than 15.0%. The fluorometric method was tested using the samples from the Jiaozhou Bay and the mesocosm experiments in the Xiaomai Island Bay in August 2007. The discrimination results of the dominant algae groups agreed with microscopy cell counts, as well as the subdominant algae groups of which the estimated relative abundance was above 15.0%. This technique would be of great aid when low-cost and rapid analysis is needed for samples in a large batch. The fluorometric technique has the ability to correctly identify dominant species with proper abundance both in vivo and in situ.