A new double-taper microfiber Mach-Zehnder interferometer (MZI) biosensor is applied for Staphylococcus aureus (S. aureus) detection. The microfiber MZI structure is fabricated by creating two tapers along a traditional single mode fiber (SMF) firstly and tapering the SMF sandwiched between two tapers into very small diameter (in the order of micrometers). The measured refractive index (RI) sensitivity of the microfiber MZI is up to 2731.1 nm/RIU in the RI range of 1.34 when the taper waist diameter was 10.2 μm, which is in good agreement with numerical simulation results by using the beam propagation method (BPM). The microfiber MZI functionalized with pig immunoglobulin (pig IgG) could be used to specifically binding to S. aureus. In experiment, the maximum wavelength shift of 1.408 nm was achieved when the microfiber biosensors were immersed into S. aureus with concentration of 7×101 CFU/mL. The limit of detection (LoD) of the microfiber biosensor for S. aureus is calculated as low as 11 CFU/mL. The proposed microfiber MZI biosensor has advantages of simple structure configuration, high sensitivity, good repeatability and specificity, wide detection range and fast detection response time (<30 minutes) and thus was demonstrated a good application prospect in food safety inspection, biochemical sensing, diseases and medical diagnostics.