A novel sulfoalkylbetaine-based zwitterionic organic-silica hybrid monolith was synthesized by using 3-dimethyl-(3-(N-methacrylamido) propyl) ammonium propane sulfonate (DMMPPS, neutral sulfoalkyl-betaine monomer). The added amount of zwitterionic monomer was significantly increased when DMMPPS was used instead of the conventionally used acidic sulfoalkyl-betaine monomer, that is, the N,N-dimethyl-N- methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine, and this led to a significantly improved hydrophilicity of the monolith. The DMMPPS-based organic-silica hybrid monolith exhibited good mechanical stability and excellent separation performance. About approximate to 20 m plate height (corresponding to column efficiency of approximate to 50000 plates/m) was obtained for nucleoside at the linear velocity of 1 mm/s. The proposed monolithic column was successfully applied to separate purines/pyrimidines, nucleotides, and tryptic digest of bovine hemoglobin in a nano-HILIC mode, and the results demonstrated that such monolith has the potential for separation of a variety of hydrophilic substances.