It has been reported that nanoplastics (NP) could cause serious toxicity and accumulative effects on aquatic organisms as well as interact with organic pollutants and influence potential hazards when exposed to biota. The current study aimed to quantitatively investigate the combined acute toxic effect of polychlorinated biphenyls (PCBs) and nano sized polystyrene (PS) plastic on aquatic organisms based on analyte speciation. First, the combined acute toxicity of PCB-18 and 100 nm PS to Daphnia magna (D. magna) in water was evaluated. Then, speciation analysis of the exposure system was conducted by measuring the sorption coefficients (logK(Np)) of PCBs to nano-sized PS (ranging from 5.28 to 6.56), which demonstrated the PS could substantially decreacP the free concentrations of PCBs. The results showed that a low concentration of the PS could decrease the toxicity to D. magna, which might be originated from the decreased free concentration of PCB-18. However, when the PS concentration was high enough, an opposite effect was observed because the PS dominated the lethality instead of PCB-18. The current study is helpful to clarify the PCB occurrence in ecosystems and provide an in-depth understanding of the eco-toxicological effects of nanoscale plastics.