Anaerobic digestion (AD) is an effective and economical in secondary sludge management. Thermal hydrolysis process (THP) is a reliable pre-treatment method to improve the biodegradability of sludge. Partial nitritation and anammox process (PN/A) is a suitable method used to remove nitrogen in reject water which has a low COD/N ratio. However, some organic compounds produced in THP process, such as melanoidins, have negative effects on AOO and anammox bacteria. Considering that humic substances occupy a large part of the soluble organics in reject water of THP-AD process. Therefore, this study aimed to investigate the acute and long-term toxicity of three types of humic substances (commercial humic acid, melanoidins and reject water) on partial nitritation step of PN/A process. The biodegradability of three types of humic substances under aerobic and anaerobic conditions, and the evolution of humic substances during the biodegradability assays were also analysed. The results showed that all three types of humic substances had higher biodegradability under aerobic condition than that under aerobic condition, and melanoidins had more biodegradable compounds than reject water and humic acid. During the AD process, humic substances acted as electron acceptors, decreasing the cumulative methane production of all three types of humic substances compared to the blank. Also, small MW fractions were transformed to large MW fractions in all three types of humic substances during both aerobic and anaerobic digestion. Moreover, during aerobic and AD, both fulvic acid-like and humic acid-like substances were formed. In addition, the results of acute toxicity tests showed commercial humic acids had a stronger inhibitory effect than reject water and melanoidins on AOO biomass. In addition, a positive correlation between humic substances concentration and AOO activity loss was observed. The long-term operation of PN reactor was failed due to the formation of granular sludge in the reactor and the growth of AOO biomass in the tubing and culture medium.