Osteosarcoma (OS), the most prevalent primary bone malignancy in adolescents, is typically treated with cisplatin-based chemotherapy. However, the development of cisplatin resistance often leads to relapse or metastasis, significantly impairing therapeutic efficacy. To tackle this issue, patient-derived osteosarcoma organoids (OSOs) is established that accurately reflect the cellular composition and heterogeneity of the original tumors, as validated by single-cell RNA sequencing, bulk RNA sequencing, and histopathology analysis. Cisplatin resistance is successfully induced in these OSOs, creating a clinically relevant model for investigating chemoresistance. Utilizing RNA sequencing in cisplatin-resistance OSOs and CRISPR screening in OS cell line, ERCC6 is identified as a pivotal regulator of cisplatin resistance. Knockdown of ERCC6 markedly enhanced cisplatin sensitivity in vitro and in vivo. Mechanistically, ERCC6 interacts with HNRNPM, influencing the PI3K/AKT signaling pathway and alternative splicing of pre-mRNA for BAX. Notably, the knockdown of ERCC6 and HNRNPM increased expression of full-length BAX and reduced skipping of exon 2, thus promoting apoptosis. This exon skipping in BAX results in a frameshift and introduces a premature stop codon (TGA) within the BH3 domain. These findings underscore the utility of OSOs in elucidating resistance mechanisms and highlight ERCC6 and HNRNPM as potential therapeutic targets.