The enrichment of co-reactants is one of the keys to improving the sensitivity of electrochemiluminescence (ECL) detection. This work developed a novel hydrophobic localized enrichment strategy of co-reactants utilizing the inner hydrophobic cavity of β-cyclodextrin (β-CD). Pt nanoparticles (Pt NPs) were grown in situ on the coordination sites for metal ions of β-CD to prepare the β-CD-Pt nanocomposite, which could not only enrich co-reactant 3-(dibutylamino) propylamine (TDBA) highly efficiently through its hydrophobic cavity but also immobilize TDBA via the Pt-N bond. Meanwhile, the carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] (PDP) polymer nanoparticles (PNPs) were developed as excellent ECL luminophores. With SARS-CoV-2 nucleocapsid protein (ncovNP) as a model protein, the TDBA-β-CD-Pt nanocomposite combined PDP PNPs to construct a biosensor for ncovNP determination. The PDP PNPs were modified onto the surface of a glassy carbon electrode (GCE) to capture the first antibody (Ab1) and further capture antigen and secondary antibody complexes (TDBA-β-CD-Pt@Ab2). The resultant biosensor with a sandwich structure achieved a highly sensitive detection of ncovNP with a detection limit of 22 fg/mL. TDBA-β-CD-Pt shared with an inspiration in hydrophobic localized enrichment of co-reactants for improving the sensitivity of ECL detection. The luminophore PDP PNPs integrated TDBA-β-CD-Pt to provide a promising and sensitive ECL platform, offering a new method for ncovNP detection.