To investigate the compositional dissolved organic matter (DOM) structure as a function of molecular weight (MW), high performance liquid chromatography (HPLC)-size exclusion chromatography (SEC) with sequential on-line detectors consisting of UV, fluorescence, and DOC was applied. Fluorescence excitation-emission matrix (EEM) spectrophotometry was also used to select wavelengths for the HPSEC on-line fluorescence system. The chosen peak maxima locations of excitation-emission wavelengths were 278 nm-353 nm for protein-like substance and 337 nm-423 nm for fulvic-like substance through an analysis of various sample and reference fluorescence EEM spectra. This system provides quantitative and qualitative information on the specific MW of DOM components regarding DOC concentration (by DOC detection), aromaticity (by comparison of UV and DOC detection), and chemical properties (by fluorescence detection). It further allows distinguishing organic matter components (e.g., fulvic-like, protein-like, and polysaccharide-like substances) as a function of MW. Three types of samples (Irvine Ranch groundwater (IRWD-GW), Barr Lake surface water (BL-SW), and secondary effluent of Hawaiian wastewater) were analyzed by the HPSEC-UVA-fluorescence-DOC system. The results of IRWD-GW were compared with fluorescence EEM spectrophotometry of samples fractionated by HPLC-SEC. The organic matter over a high MW range (over 10,000 g/mol) consisted of polysaccharide-like substances for IRWD-GW and a mixture of polysaccharide-like/protein-like substances for BL-SW and the wastewater secondary effluent. Minimal amounts of fulvic-like substances were found in the wastewater secondary effluent sample. The fractions over a medium MW range (5,000-1,000 g/mol) showed higher aromaticity (humic in character) than any other fractions for all samples. Over a low MW range (below 680 g/mol), additional aliphatic organic matter was found in IRWD-GW while BL-SW contained a protein-like substance. DOM plays an important role in water treatment and wastewater treatment processes. An enhanced HPSEC technique with multiple on-line detectors enables a better understanding of quantitative and qualitative properties in water or wastewater samples and helps to design and optimize water/wastewater treatment facilities. Includes 15 references, tables, figures.