Remote sensing system, especially hyperspectral remote sensing require fundamental knowledge of spectral reflectance of chlorophyll a (chl a) for recognizing eutrophication of inland waters. The first objective of this study was to prepare and investigate the significant differences between the spectral signature of water samples with different amount of chlorophyll a (chl a) of Anzali wetland in 15 cm depth. This was carried out using a full range spectrometer during the spring 2013. The second objective of this study was to discriminate the spectral signature of water samples with different amount of chlorophyll a (chl a) of Anzali wetland in 30 cm depth. A total of 500 water sample spectral curves of illuminated and shaded samples were acquired of 80 water samples with different amount of chlorophyll between 2.07 and 23.9 mg/l. Following the measurements, chlorophyll and total phosphorus of the samples were extracted in laboratory. One important index related to chlorophyll a of water was calculated and statistically analyzed. We conclude that band ratio model in 15 cm depth of water samples has the most relation with chlorophyll a content in comparison with the other indices. This result has been proved by statistical results obtained by chlorophyll and total phosphorus data in lab.