The available information about whether wastewater-derived disinfection byproducts (DBPs) could elicit potential endocrine-related detrimental effects on aquatic organisms was scarce. Herein, the potential disrupting effects and underlying binding mechanism of 14 wastewater-derived aliphatic and aromatic DBPs and 12 other substances on Chinese rare minnow (Gobiocypris rarus) transthyretin (CrmTTR) were tested and revealed by in vitro and in silico methods. The amino acid sequences of CrmTTR were determined, and the recombinant CrmTTR with a molecular mass of 66.3 kDa was expressed and purified. In vitro assay results indicated that eight selected aromatic DBPs exhibited detectable CrmTTR disrupting ability. Meanwhile, six aliphatic DBPs were not CrmTTR binders. Molecular modeling results implied that hydrophobic hydrogen bonds and/or ionic pair interactions were non-negligible. Four binary classification models with high classification performance were constructed. A significant positive linear relationship was observed for the binding affinity data from CrmTTR and human TTR (n = 18, r = 0.922, p < 0.0001). However, the binding affinity for 13 out of 18 tested compounds with CrmTTR was higher than that with human TTR. All the results highlighted that some wastewater-derived DBPs may be potential disruptors on the aquatic organism endocrine system, and interspecies variation should not be neglected in future determination of the potential endocrine disrupting effects of wastewater-derived DBPs.