The development of cost-effective, portable, and ease-of-use sensing system for on-site genetic diagnostics is highly desirable for pathogen screening and infectious disease diagnosis. This study develops (1) a paper-based biochip which is able to integrate the loop-mediated isothermal amplification (LAMP) protocols for simultaneous detection of Escherichia coli O157:H7, Salmonella spp., and Staphylococcus aureus , and (2) a stand-alone smartphone-based portable device which can control exactly 65 °C for isothermal amplification as well as collect and analyze the thus generated fluorescence signals. The reported sensing system has been successfully demonstrated for foodborne pathogen detection with a limit of detection of 2.8 × 10 −5  ng μL −1 . Spiked milk samples with concentration as low as 10 CFU mL −1 were successfully determined within 4 h, demonstrating the practicality of the reported sensing system in the fields. The reported sensing system featuring simplicity and reliability is ideally suited for genetic diagnostics in low resource settings. Graphical abstract