In this work, the wet-corrosion process for the synthesis of titanium oxide (TiO2) nanowires in the anatase phase was optimized as the solid matrix in MALDI-TOF mass spectrometry, and the solid matrix of the TiO2 nanowires was applied to the detection of antibiotics in a daily milk sample. The influence of the alkali concentration and the heat treatment temperature on the crystal structure of the TiO2 nanowires was investigated. The ionization activity of the TiO2 nanowires was estimated for each synthetic condition using amino acids as model analytes with low molecular weights. For the detection of antibiotics in milk, benzylpenicillin was spiked in daily milk samples, and MALDI-TOF mass spectrometry with the TiO2 nanowires was demonstrated to detect the benzylpenicillin at the cut-off concentration of the EU directive.