Biological surface-enhanced Raman scattering (SERS) sensing is dependent on the properties of the sensing surface. For the biological sensing surfaces for SERS, the hydrophilicity, biocompatibility and signal sensitivity are pivotal. Hence, a hard bimetallic Mo-Ag film was developed by a magnetron sputtering technique, and the surface morphology could be controlled by the relative contents of Mo and Ag. The Mo-Ag film has better hydrophilicity than the pure Ag film, which could be beneficial for cell attachment during biological SERS sensing. Moreover, the cell test shows that the Mo-Ag film exhibits good cytocompatibility with MC3T3-E1 cells, which shows potential for SERS detection in vivo. The high surface roughness of the Mo-Ag film is a fascinating feature for enhancing Raman scattering signals. Through depositing a thin film of Mo-Ag on a glass surface with a size of 0.5 × 0.5 cm2, a sensing chip of SERS could be produced. High sensitivity and excellent signal reproducibility were acquired. The sensitivity was down to 10-10 mol L-1 for malachite green and 10-9 mol L-1 for thiram, and the relative standard deviation value was lower than 7.0%. Moreover, bacteria were detected by employing the Mo-Ag film chip, and the difference in signal uniformity between molecules and bacteria is illustrated. In summary, depositing the Mo-Ag film on the surface of sensors could be an effective strategy for biomedical SERS sensing.