Abstract: A biosensor essentially consists of 2 main components viz., a physical transducer and a biorecognition element. In this study, a magnetostrictive platform is served as the transducer, and as the mass sensitivity, the magnetoelastic resonance sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied time-varying magnetic field. Due to the magnetoelastic nature of the amorphous magnetostrictive alloy, the sensor exhibits a physical resonance when it undergoes a time-varying magnetic field, and a shift in resonance frequency of the magnetostrictive sensor depends only on the mass change when environmental parameters are invariable. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that its measurement is wireless and remote. And phage, which has been verified to be thermally stable, is used as the biorecognition element. In this paper, a multiple phage-based magnetoelastic (ME) biosenor system for simultaneously detecting Salmonella typhimurium and Bacillus anthracis spores was prepared by immobilizing 2 different kinds of phages as biorecognition element onto the magnetoelastic thin film made from 2826 MB MetglasTM, and the 2 kinds of phages were the E2 phage specific to Salmonella typhimurium and the JRB7 phage specific to Bacillus anthracis spores, respectively. Finally, 1 mg/mL bovine serum albumin (BSA) was immobilized onto the magnetoelastic thin film as blocking agent for getting specific binding of target bacteria. The multiple phage-based magnetoelastic (ME) biosensor system was simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to Salmonella typhimurium, and a JRB7 phage-coated sensor specific to Bacillus anthracis spores. The sensors were free standing during the test, and held in place by a magnetic field. In the detection process, the environment parameters were kept constant, and the changes in the resonance frequency of the biosensors, which were recorded by HP network analyzer 8751A over the testing period, were attributed only to the binding of the phages with target analyte of Salmonella typhimurium or Bacillus anthracis spores; the binding was also visually confirmed by the scanning electron microscopy (SEM) micrographs by observing the surface of each kind of biosensor, and there were up to 10 different regions on each sensor surface which were examined and photographed by the SEM to obtain the reliable and statistical data. According to the shift in resonance frequency due to the binding of the phages with Salmonella typhimurium or Bacillus anthracis spores, the binding specificity and sensitivity of the biosensor were evaluated. The detection results showed that after sequential exposure to pathogenic solutions individually, only the biosensor coated with the corresponding specific biorecognition element of phage had the response. As the cells or spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking agent and verified by the reference sensor, and the SEM measurement showed there was no frequency shift after the reference sensor was immersed in the solution for the same duration. The detection results also demonstrated that the multiple magnetoelastic sensors could be used simultaneously to detect specifically targeted pathogenic species and had good selectivity. The results show that the developed magnetostrictive biosensors can be applied for detecting Salmonella typhimurium and Bacillus anthracis spores simultaneously.