Phytoplankton are crucial producers in marine ecosystems. The discrete three-dimensional fluorescence spectroscopy method addresses the high operational requirements of continuous three-dimensional fluorescence spectrophotometers, thus meeting the needs for online and in-situ phytoplankton monitoring. However, challenges persist due to the instability of living fluorescence and the similarity in pigment composition among different algal species. This reduces the spectral differentiation between algal groups in discrete three-dimensional fluorescence spectroscopy and limits its practical application. This study investigates the three-dimensional fluorescence spectral characteristics of Bacillariophyta and Dinophyta under various environmental conditions. It analyzes the limitations of the commonly used average concentration-normalized standardized spectral library construction methods (hereinafter referred to as the “average method”). Based on this, a standardized phytoplankton spectral library construction method is proposed using uniform interpolation (hereinafter referred to as the “interpolation method”). The optimal interpolation range and number of interpolations for the phytoplankton standardized spectral library were determined through experiments. The results show that an average coverage rate of 88% and an average correlation coefficient of 0.98 are achieved within the range of M±2S, which is identified as the optimal interpolation range. With 10 interpolations, measurement accuracy improves by nearly 13% compared to the average method, and the computation time is only 0.494 8 seconds, marking it as the optimal number of interpolations considering both accuracy and computational efficiency. Accordingly, a standardized spectral library for Bacillariophyta and Dinophyta was constructed based on uniform interpolation. Combined with non-negative least squares linear regression analysis, this method was used to interpret the three-dimensional fluorescence spectra of a series of algal samples with known concentrations, and the interpretation results were compared with those obtained using the average method. The results indicated that the interpolation method showed a correlation coefficient (k value) ranging from 0.828 to 1.149 and a determination coefficient (R²) ranging from 0.616 to 0.953 for five pure algal samples. The number of Bacillariophyta samples that failed to be identified decreased significantly from 21 (with the average method) to only 2, and the average absolute relative errors in the analysis of Bacillariophyta and Dinophyta were 36.9% and 30.7%, respectively, representing reductions of 20.6% and 19.0% compared to the average method. These results indicate the effectiveness of the uniform interpolation-based method in improving the accuracy of quantitative analysis of Bacillariophyta and Dinophyta. After validating the method's effectiveness in the laboratory, this uniform interpolation-based standardized spectral library construction method was extended to Cyanophyta, Chlorophyta, and Cryptophyta and applied to AFA during a cruise in the South China Sea. This research provides an effective technical method for the rapid and accurate classification and measurement of phytoplankton, offering strong scientific support for future marine ecological monitoring and environmental protection.