Serving as a representative indicator for phytoplankton and water quality monitoring, Chlorophyll a (Chl-a) is of great significance to evaluating lake eutrophication level. In order to explore the hyperspectral characteristics of multi-temporal Chl-a concentration and to select the best inversion methods of Nanyi Lake, 98 sets of hyperspectral data and Chl-a concentration data were collected simultaneously from 8 navigational water experiments in Nanyi Lake from 2020 to 2021 were selected. To extract the characteristic bands most sensitive to Chl-a concentration, measured spectrum data of Nanyi Lake under different Chl-a concentration levels were analyzed, considering the influence of changes in water quality at different timeson the spectrum. Then, the peak and valley distance method, the fluorescence line height method, the Normalized peak area method and the peak area above valley method was introduced to jointly invert the concentration of Chl-a in Nanyi Lake, followed by inter-comparing the results of the abovementioned algorithms based on the 5-fold cross-validation. The results areas follows: (1) As the concentration of Chl-a increases, the absorption valley and fluorescence peak of Chl-a tend to deepen and increase, respectively. At the same time, the position of the fluorescence peak moves towards the infrared part with increasing Chl-a concentration. The obvious difference between peak and valley under different Chl-a concentration levels indicates spectrum before and after fluorescence peak is highly sensitive to the change of Chl-a concentration. (2) Validation results using a 5-fold cross-validation method show that the mean values of RMSE and MAPE extreme differences for each method for different groups of validation sets were 0.437 5 μg·L-1 and 28.27%. It can be seen that the sampling method of the modeling set and verification set will introduce evaluation error, which can effectively be reduced by the 5-fold cross-validation method, obtaining the pros and cons of each method to the greatest extent based on samples. (3) Best inversion results have been achieved by the peak area above valley method, which was proposed in combination with the horizontal tangent line at the minimum value of the absorption valley of Chl-a concentration, with R2=0.756 7, RMSE=1.653 1 μg·L-1, and MAPE=40.77%. Compared with the peak and valley distance method, the fluorescence line height method and the Normalized peak area method witnessed significant improvement in the inversion accuracy and provided a new idea for the inversion of chlorophyll concentration based on fluorescence.