Revealing multiple biomolecules in the physiopathological environment simultaneously is crucial in biological and biomedical research. Mass spectrometry (MS) features unique technical advantages in multiplexed and label-free analyses. However, owing to comparably low abundance and poor ionization efficiency of target biomolecules, direct MS profiling of these biological species in vitro or in situ remains a challenge. An emerging route to solve this issue is to devise mass tag (MT)-encoded nanointerfaces which specifically convert the abundance or activity of biomolecules into amplified ion signals of mass tags, offering an ideal strategy for synchronous MS assaying and mapping of multiple targets in biofluids, cells and tissues. This review provides a thorough and organized overview of recent advances in MT-encoded nanointerfaces elaborately tailored for several practical applications in multiplexed MS bioanalysis and biomedical research. First, we start with elucidation of the structural characteristics and working principle of MT-encoded nanointerfaces in specific labeling and sensing of multiple biological targets. In addition, we further discuss the application scenarios of MT-encoded nanointerfaces particularly in multiplexed biomarker assays, cell analysis, and tissue imaging. Finally, the current challenges are pointed out and future prospects of these nanointerfaces in MS analysis are forecast.