Mass spectrometry (MS) based analysis has received intense attention in diverse biological fields. However, direct MS interrogation of target biomolecules in complex biological samples is still challenging, due to the extremely low abundance and poor ionization potency of target biological species. Innovations in nanomaterials create new auxiliary tools for deep and comprehensive MS characterization of biomolecules. More recently, growing research interest has been directed to the compositional and structural engineering of nanomaterials for enriching target biomolecules prior to MS analysis, enhancing the ionization efficiency in MS detection and designing biosensing nanoprobes in sensitive MS readout. In this review, we mainly focus on the recent advances in the engineering of nanomaterials towards their applications in sample pre-treatment, desorption/ionization matrices and ion signal amplification for MS profiling of biomolecules. This review will provide a toolbox of nanomaterials for researchers devoted to developing analytical methods and practical applications in the biological MS field.