Humic substances (HS) are formed in estuarine regions by biogeochemical processes of terrestrial and in situ produced organic matters. Their structure and distribution may vary seasonally and spatially. To examine this, HS were isolated from Godavari estuarine waters using ion-exchange resins XAD-8 followed by XAD-4 during 2014-2015. The structural differences between the two fractions were characterized by E 2 /E 3 ratio (a 250 /a 365 ), spectral slope (S 275-295 ), and spectral slope ratio (S R , S 275-295 /S 350-400 ), which were derived from UV-visible absorbance spectra. Lower values of E 2 /E 3 ratio, S 275-295 and S R for XAD-8 fractions than XAD-4, indicate higher aromaticity and higher molecular weight of dissolved organic compounds retained on the former resin. The E 2 /E 3 ratio for XAD-8 and S R for XAD-4 fractions were found to decrease gradually from post-monsoon to monsoon, indicating that the biological process controls the production of organic matter in upstream waters. Lower molecular weight organic compounds formed by bacterial decay and photodegradation during pre-monsoon and higher molecular weight organic compounds formed during the post-monsoon season was attributed to the freshly exudated organic matter from phytoplanktons dominated at the mouth of the estuary.