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[Display omitted] • SUVA 254 and HIX are indicators for bioavailability. • Dissolved organic matter over 10 kDa transformed into 0–10 kDa, which improved bioavailability. • The protein-like matter of 0–5 kDa is critical for improving bioavailability. • The humic-like matter of 5–100 kDa is essential for improving bioavailability. In this study, swine wastewater (SW) and cow wastewater (CW) were used for anaerobic digestion (AD). We found the bioavailability of dissolved organic matter (DOM) was affected by the molecular weight ranges and molecular composition during the AD process. The organic substance in the small molecular range (0–5 kDa) accumulated due to a larger molecular fraction (>10 kDa) degradation, which enhanced the bioavailability of the DOM. Moreover, based on the excitation emission matrix-parallel factor (EEM-PARAFAC) analysis, the protein-like component in 0–5 kDa molecular size and humic-like component over 5 kDa are significantly positively correlated with DOM bioavailability. This study indicated that increasing the hydrolysis of larger organic matter and humification degree of molecular weights>5 kDa are critical solutions to improving the bioavailability of DOM. These conclusions can help explain the molecular mechanisms of DOM transformation and the AD process of livestock wastewater. [ABSTRACT FROM AUTHOR]