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Mass cytometry (MC) is an emerging and powerful bioanalytical technique for high-dimensional single-cell analysis. While metal-chelating polymers (MCPs) have been the most successful mass tag reagents for MC, nanoparticle (NP)-based mass tag reagents are of great interest to improve the sensitivity of MC toward low-abundance biomarkers. Here we present a new structure design for potential MC probes using multifunctionalized mesoporous silica nanoparticles (MSNs), modified with zwitterionic sulfobetaine silanes and long-chain polyethylene glycol silanes (PEG5k, M= 5000). The resulting methoxy-terminated NPs (PMSN-Zwi-mPEG5k) displayed uniform size, good redispersibility and colloidal stability, as well as versatility in accommodating 13 types of lanthanides. They exhibited the capacity to carry up to 7.4 × 104Tb ions per NP, with negligible ion loss observed in both H2O and 1× PBS buffer. We investigated the interactions of these functionalized NPs with serum proteins using UV–vis, and with peripheral blood mononuclear cells (PBMCs) using MC. By varying PEG5kchain density on NP surface, we could minimize their nonspecific binding (NSB) to human serum albumin proteins, while also reducing their NSB to PBMCs at the titer of 1000 NPs/cell. Additionally, unconjugated NPs showed good compatibility with commercial Maxpar MCP mass tags in a 10-plex assay for PBMCs staining. Under these optimized conditions, N3-terminated NPs (PMSN-Zwi-PEG5k-N3) were synthesized and conjugated with antibiotin antibodies (Abs), resulting in effective binding of biotin Cy5 molecules. We finally compared eight different bioconjugation conditions to maximize bioconjugation efficiency with retention of Ab function. These preliminary findings demonstrate the fundamental technical capabilities and promising prospects for the future application of MSN-based mass tag reagents in MC.