The rapid identification of bacteria is of increasing interest in food, biosafety, and clinical analysis. In this paper, we report a technique for rapid lipid profiling of bacteria by online droplet-deposited MALDI mass spectrometry. In the experiment, lipids in bacterial cells (Escherichia coli, Salmonella, and Bacillus subtilis) are extracted with the mixture of chloroform/methanol via ultrasonication. The extracts are nebulized into suspended droplets and then instantly analyzed with an online droplet-deposited MALDI-TOF mass spectrometer. The lipid profiles of the three bacteria cultured in nutrient broth and LB (Lysogeny Broth) medium are obtained. The lipids phosphatidylethanolamine (PE), diglycosyl diacylglycerol (DGDG), and phosphatidylglycerol (PG) are observed in the MALDI-TOF mass spectra. The MALDI-TOF mass spectrum of each bacterial extract shows a characteristic profile. The experimental results demonstrate that the present technique is capable of conducting rapid lipid analysis and has the potential for rapid bioaerosol detection.