Abstract: Exogenous virus contamination represents a critical risk to biopharmaceutical safety. Conventional detection methods, constrained by insufficient sensitivity, limited broad-spectrum capability, and reliance on animal experimentation, struggle to address the screening demands for low-abundance or unknown pathogens. Metagenomic next-generation sequencing(mNGS), with its high sensitivity and unbiased nature, offers a novel solution for exogenous virus detection and has been endorsed as a recommended method in ICH Q5A(R2). However, mNGS faces multiple technical challenges in practical applications: host-derived nucleic acid interference(>99% of total content) obscures target signals, reagent contamination and index hopping introduce false-positive risks, incomplete databases and controversies over analytical algorithm thresholds exacerbate misidentification of unknown pathogens, and the lack of standardized protocols compromises inter-laboratory comparability. Through case studies, this paper comprehensively analyzes the challenges encountered in mNGS implementation and proposes targeted optimization strategies, including host nucleic acid depletion, unique dual indexing, and multicenter standardization validation. To advance mNGS as a “regulatory-grade method”, collaborative efforts in technological innovation, unified pathogen databases, and regulatory harmonization are essential to establish robust technical frameworks for biopharmaceutical safety evaluation.