Abstract: Magnetostrictive biosensors can often consist of magnetostrictive transducers, functional coatings, signal excitation, bioprobes, and detection systems. These sensors can be used to generate and transmit the signals via magnetic field coupling. Among them, the magnetostrictive material can be subjected to the excitation of alternating magnetic fields during detection. Once the frequency of the magnetic field matches the natural frequency of sensors, the resonance can occur for an impedance signal. A direct current (DC) bias magnetic field can also be applied to amplify the signal. At the same time, the adsorption of pathogens can increase the mass of the sensor, leading to the variation in the resonance frequency. Some key factors (such as manufacturing costs, detection sensitivity, and efficiency) can significantly influence the performance of magnetostrictive biosensors. In this study, a series-connected magnetostrictive biosensor was developed to integrate two sensors with different sizes. A Fe-based amorphous soft magnetic alloy was selected with 25 µm thick as the transducer. The functional coating with a 350 nm thick was prepared as the polyvinyl alcohol (PVA) film from a 2.5% PVA solution. Single sensors were used with the dimensions of 3.6×0.6, 4.8×0.8, 6.0×1.0, 7.2×1.2, and 8.4 mm×1.4 mm as the control groups. Series connections were made as follows: 3.6×0.6 with 6.0×1.0, 4.8×0.8 with 7.2×1.2, and 6.0× 1.0 with 8.4 mm×1.4 mm. An impedance analyzer was applied with a 50 mV alternating current (AC) excitation and a 6V DC bias voltage. A comparative analysis was conducted on the resonance parameters of single- and series-connected sensors. The series-connected sensors were selected with the dimensions of 4.8 mm×0.8 and 7.2×1.2 mm to detect Escherichia coli (E. coli) suspensions with concentrations ranging from 10 to 10⁸ CFU/mL with/without antibodies. The adsorption of pathogenic bacteria was analyzed on some shifts in resonance frequencies. Scanning electron microscopy (SEM) was used to observe the surface adsorption of pathogenic bacteria on sensors. A systematic investigation was made to explore the relationship between the resonance frequency shifts and immersion times when the E. coli was detected in the food solutions using series-connected magnetostrictive sensors. The results revealed that there was a close relationship between the resonance parameters and the size of single- and series-connected sensors. The resonance frequency decreased, as the sensor size increased, whereas, the resonance amplitude increased. The magnitude of the resonance amplitude was quantitatively analyzed using the resonance frequency. Additionally, the series-connected magnetostrictive biosensors demonstrated that there was nearly unchanged in the resonance frequencies, but there was an increase in the resonance amplitudes, compared with the single sensors. Therefore, an enhanced strength response was achieved as well. The frequency shift of resonance increased with the higher concentrations of E. coli suspensions. Antibody-loaded sensors exhibited significantly larger resonance frequency shifts than those without antibodies, due to the specific binding between antibodies and E. coli. The detection limit of the series-connected sensor was determined to be 10² CFU/mL. There were relatively close frequency shifts of resonance in the 4.8 mm× 0.8 mm and the 7.2 mm×1.2 mm sensors in the same loading conditions, due to the higher number of bacteria that adsorbed by the larger sensor (7.2 mm×1.2 mm). The small difference in mass between the two sensors resulted in similar frequency shifts. SEM observations confirmed that the frequency shifts of resonance were caused by the adsorption of E. coli. The sensor demonstrated excellent detection. The sensor with the antibody loading adsorbed significantly more E. coli on the surface, compared with the sensor without antibodies. This finding was consistent with the measurements on the frequency shift of resonance. The sensor achieved stable detection within 20 minutes. The novel series-connected magnetostrictive biosensor can offer high sensitivity, strong signal strength, high detection efficiency, simple manufacturing, low cost, and ease of use. This finding can provide a strong reference to apply biosensors to safety detections in the food industry.