The present research was based on the single drop microextraction (SDME) approach using an ionic liquid drop to extract bacteria from aqueous samples for characterization of pathogenic bacteria by MALDI-TOF MS. The SDME of bacteria from aqueous samples was successfully achieved by using platinum nanoparticles mixed in ionic liquid (IL, 1-butyl-3-methylimidazolium hexafluorophosphate) and IL alone as the extraction drops. The IL was used as liquid drops by SDME to obtain protein profiles from bacteria. Significant numbers of biomarker protein peaks of bacteria were identified from target biological samples. The IL also significantly improved the signal reproducibility of spectra using the SDME approach combined with MALDI-TOF MS. Thus, the present technique was successfully applied to detect pathogenic bacteria at low concentrations of 10⁶ cfu mL⁻¹ from aqueous suspensions. The selected bacteria viz., Escherichia coli and Serratia marcescens were used as target biological sample.