Exosomes contain abundant molecular information derived from parental cells and represent promising biomarkers for liquid biopsy. However, current exosome detection methods often suffer from complicated procedures, insufficient sensitivity, and limited applicability in complex biological samples. Herein, we developed a nanopore-based enzyme-linked immunosorbent assay (NELISA) for sensitive exosome analysis. This strategy employs exosomal surface antigens to construct alkaline phosphatase (ALP)-labelled sandwich immunocomplexes, followed by enzymatic conversion of the phosphorylated peptide probe FGpYD₈ (where “p” denotes phosphorylation modification). The substrate and product peptides generate distinct current signatures during translocation through a protein nanopore, enabling quantitative analysis through statistical evaluation of nanopore events. By rational selection of capture antibodies, the proposed method enables differential analysis of exosomes from distinct cellular origins and achieves a broad dynamic range of 10²–10⁹ particles/mL with a detection limit of 10² particles/mL for HeLa cell-derived exosomes, while maintaining reliable performance in complex biological matrices. This work provides a sensitive and versatile platform for exosome analysis and highlights its potential for exosome-based liquid biopsy applications.