Herein, we proposed an integrated platform combining organoid modeling with longitudinal EV proteomics to accelerate dynamic biomarker discovery, addressing the problem of costly and time-consuming detection of lymphatic metastasis in heterogeneous BC progression. And for precise EV proteomic analysis of individual organoids, all organoids were cultured separately and analyzed. By using ultra-sensitive mass spectrometry method for their EV proteomics, we obtained time-resolved, in-depth EV proteomic profiling that captures evolving molecular signatures during organoid progression, and multi-level clinical validation emphasizes the diagnostic potential of screened markers by using matched tissue and plasma.