The influence of dissolved organic matter (DOM) on mineral extraction from salt lake brines depend on DOM quality. This study contributes to our knowledge of DOM’s metal binding behavior in hypersaline environments by characterization of DOM from lakes in the Qaidam Basin, i.e., Qarhan Lake (LQDOM), Da Qaidam (DQDOM) and West Ginair Salt Lake (WGDOM). The DOM was fractionated based on solid phase extraction (SPE) and ultrafiltration (UF), and the spectral and metal binding behavior of these fractions were studied by absorption spectroscopy, Pb(II) titration techniques and fluorescence parallel factor (PARAFAC) analysis. The results showed that bulk DOM generally contained more dissolved organic carbon (DOC), lower specific UV absorbance (SUVA254), higher fluorescence and biological indices, comparable humification index, and lower condition stability constants compared to the other nature waters. Compared with UF, SPE-derived DOM exhibited higher DOC recovery and aromaticity and lower carbohydrate yield. It appears that the SPE procedure used affects the spectral composition of bulk DOM to a larger extent than UF. Source and molecular weight (MW)-dependent differences in abundance and quality of brine DOM was indicated by higher SUVA254 in high MW DOM, for LQDOM and DQDOM, and humic-like fluorophores were mainly in high MW-DOM in each lake. Moreover, the high MW humic-like component exhibited higher metal binding potential than the bulk and low MW counterparts for LQDOM and DQDOM, while the inverse was observed for WGDOM. This study revealed the effects of isolation techniques on interpretation of DOM characteristics, and meanwhile highlighted the importance of origin- and MW-dependent DOM in manipulating the behavior, fate, and bioavailability of heavy metals in salt lake brine.