A novel ratiometric fluorescent immunoassay was developed based on silver nanoparticles (AgNPs) for the sensitive determination of dibutyl phthalate (DBP). In the detection system, AgNPs were labeled on the secondary antibody (AgNPs@Ab₂) for signal amplification, which aimed to regulate the H₂O₂ concentrations. When AgNPs-Ab₂ and antigen–primary antibody (Ab₁) were linked by specific recognition, the blue fluorescence of Scopoletin (SC) could be effectively quenched by the H₂O₂ added while the red fluorescence of Amplex Red (AR) was generated. Under the optimized conditions, the calculated detection of limit (LOD, 90% inhibition) reached 0.86 ng/mL with a wide linear range of 2.31–66.84 ng/mL, which was approximately eleven times lower than that by HRP-based traditional ELISA with the same antibody. Meanwhile, it could improve the inherent built-in rectification to the environment by the combination of the dual-output ratiometric fluorescence assays with ELISA, which also enhanced the accuracy and precision (recoveries, 87.20–106.62%; CV, 2.57–6.54%), indicating it can be applied to investigate the concentration of DBP in water samples.