Abstract For the first time, we report the fabrication of a titanium bacterial chip for MALDI-MS produced from a simple, cost effective and rapid heat treatment process. This bacterial chip can be reused many times and is highly versatile. These bacterial chips serve dual roles: (1) They can be applied as MALDI-MS target plates for direct and highly sensitive bacterial analysis. (2) They can be used as bacterial sensors for direct analysis of the captured bacteria using MALDI-MS. The sensitivity of these chips when used as bacterial sensors is <103cfu/mL. The lowest detectable concentration for direct MALDI-MS analysis was found to be 104cfu/mL. The results were further justified by using standard plate counting method combined with Tukey–Kramer statistical analysis and fluorescence imaging followed by image processing for fluorescence quantification using ImageJ software to substantiate the MALDI-MS results.