The rapid detection of Staphylococcus aureus (S. aureus), particularly in the case of very low levels of S. aureus, is very important in food safety and medical diagnostics. In this paper, a fast (<20 mins), label-free biosensor using a U-shaped single mode- tapered multimode- single mode (STMS) fiber structure is presented. The average wavelength shift of the sensor (modified with the IgG from porcine serum with a concentration 200 μg/mL) is as high as 1.552 nm when it is immersed into an S. aureus sample with a concentration of 4 cells/mL. By further diluting the S. aureus to 0.4 cells/mL for a sample volume of 1.5 mL, it is found that a dip wavelength shift of 0.348 nm is observed on occasion which confirms that the biosensor can detect a single colony (cell) of S. aureus. Results for the detection of S. aureus in milk and lettuce samples are also presented and are compared with the wavelength shift results in phosphate buffered saline (PBS), and it is found that the variation in the average wavelength shift is − 21% for S. aureus concentrations from 4 to 4 × 103 cells/mL.