Humic acids (HAs) were extracted from sediments of different segments in the Jialingjiang River. Fluorescence spectroscopy, elemental analysis and Fourier transform infrared (FTIR) spectrometry were used to investigate the chemical composition and structure characteristics of HAs. Elemental analysis showed that H/C, O/C, and C/N molar rations were significantly different among 4 HAs, which indicated their differences in chemical composition and structure characteristics. FTIR spectra revealed that HAs contained more carboxyl, hydroxyl and carbonyl functional groups, and aromatic structure was very obvious. The three dimensional fluorescence excitation-emission-matrix (3DEEM) spectra of HAs presented two main peaks (peak a and peak b) at Ex/Em=270/390 nm and Ex/Em=230/350 nm. The relative fluorescence intensities Ia/Ib varied from 1.31 to 2.53 for different HA samples, which revealed that they contained multiple molecule fluorophores. Fluorescence index (f450/500=1.43～1.53) suggested that HAs mainly originated from terrestrial sources.