Abstract: Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has been widely used in the testing of biological samples. It will become a new path for medical microbiology applications because of its fast, accurate and high-throughput testing characteristics. At present, the MALDI-TOF MS target plates where have been widely used in medical microbiological detection are reusable steel based. This target plates have the problems of cross contamination and insufficient resolution. Therefore, based on new low-cost materials of high-performance disposable targets become a research highlight. This article introduced a new dielectric target structures and the preparation route based on low-cost glass substrate. In this study, the glass was used as the substrate to make the target plates, and its processing technology was carefully studied. The pit structure of about 6-7 μm in the sample loading area was specially designed to increase the capacity of the sample loading point, thereby improving the spotting experience. In order to produce this kind of micro-nano structure, this research chose the micro-nano processing scheme of wet etching. The wet etching process of B270 and float glass were tested successively, the etching parameters were found out, and finally a qualified target with a pattern inaccuracy of 3.8‰ was obtained. At the same time, since the target substrate was converted from conductive metal to dielectric glass, the target design had further studied the ionization performance of the test objects. It was found that the glass target must be fully nickel-plated on the back to achieve stabilized ionization. The finite element (FE) method was used to explore the mechanism of nickel plating on the back to enhance ionization, and finally a glass target with qualified performance was successfully prepared. The performance of the glass target and the steel target were compared in detail from matrix crystallization, resolution, test quality stability, and bacterial standard test stability. The results show that the glass disposable target has better crystal uniformity than the steel target, better resolution for large-quality bacterial proteins, test stability equal to the steel target, and significantly improved stability of the bacterial detection score. Due to the use of cheap and easy-to-obtain glass substrates, it is expected that the average material cost will be controlled at RMB 10 per piece in mass production, realizing low-cost replacement of steel targets.