Conventional biochemical methods for foodborne pathogen detection are time-consuming, labor-intensive and can only be used for single type bacteria detection with one sample. There is always an urgent need for fast, accurate and easy to handle devices for identification and monitoring of multiple foodborne pathogens at the same time. Herein, we present a non-biofouling polyethylene glycol (PEG) based microfluidic chip integrated with functionalized nanoporous alumina membrane for simultaneous electrochemical detection of two types of bacteria Escherichia coli O157:H7 and Staphylococcus aureus from the mixed samples. The experimental results demonstrated the specificity for target bacteria detection and the low cross-binding of non-target bacteria. The simultaneous detection of mixed bacteria sample of E. coli O157:H7 and S. aureus was also demonstrated. This sensor has a linear detection range from 10² CFU/mL to 10⁵ CFU/mL with the limit of detection (LOD) around 10² CFU/mL.