Microfluidic-based sheath flow focusing methods have been widely used for efficiently isolating, concentrating, and detecting pathogenic bacteria for various biomedical applications due to their enhanced sensitivity and exceptional integration. However, such a microfluidic device usually needs complicated device fabrication and sample dilution, hampering the efficient and sensitive identification of target bacteria. In this study, we develop and fabricate a sheath-assisted and pneumatic-induced nano-sieve device for achieving the improved on-chip concentration and sensitive detection of (MRSA). The optimized nanochannel design with diverging configuration is beneficial to the regulation of the hydrodynamic flow while the sheath flow is focusing the sample to the confined region as expected. Per the experimental finding, a high flow ratio (sheath flow/sample flow) presents enhanced target concentration by comparing with a low flow ratio. With this setup, MRSA bacteria with an extremely low concentration of ∼100 CFU mL are successfully and sensitively detected under a fluorescence microscope, less than 30 min, demonstrating a reliable sheath-enhanced concentration and on-chip detection for target bacteria. Additionally, the theoretical model introduced here further rationalizes the working principle of our nano-sieve device, potentially guiding the optimization of next generation devices for highly sensitive and accurate on-chip bacteria detection at a much lower concentration level below 100 CFU mL.