The coadsorption of mixed anionic-nonionic surfactants, sodium dodecylbenzenesulfonate with Triton X-100, on the surface of eggshell membrane was investigated based on adsorption isotherms to improve the solid-phase extraction performance of eggshell membrane toward organic contaminants. Results showed that even though excess Triton X-100 might inhibit the adsorption of sodium dodecylbenzenesulfonate, a low dosage of Triton X-100 can significantly improve sodium dodecylbenzenesulfonate modification and enhance the extraction efficiency of eggshell membrane from 73.7 to 100.4% because of the formation of mixed hemimicelles. The highest recovery was achieved at 2:8 (Triton X-100/sodium dodecylbenzenesulfonate mass ratios), and multiple mechanisms involving π-π interactions, hydrophobic effect, and π-π electron donor-acceptor interactions contributed to the strong extraction affinity. When mixed, the Triton X-100 and sodium dodecylbenzenesulfonate modified eggshell membrane packed cartridge coupled with high-performance liquid chromatography was applied for the simultaneous determination of trace Sudan I-IV, and low detection limits (0.16-0.26 ng/L) were achieved with satisfactory linearity (R > 0.999) in 10-10 000 μg/L. For real samples, Sudan II and III in one chilli sauce sample were found at 4.3 and 1.7 μg/kg. Sudan I-IV recoveries at three spiked levels were 87.4-102.9% with precisions <6.8%. Comparison with commonly used solid-phase extraction adsorbents and methods further reflected the superiorities of the proposed adsorbent in sensitivity, retention ability, and applicability.