The method capable of simultaneously detecting multiple target bacterial pathogens is necessary and of great interest. In this research, we demonstrated our initial effort to simultaneously detect seven common foodborne bacteria by developing a straightforward upconversion fluorescence sensing approach. The fluorescent nanosensor was constructed from a designed guanidine-functionalized upconversion fluorescent nanoparticles (UCNPs@GDN), tannic acid, and hydrogen peroxide (HP) and could quantify pathogenic bacteria in a nonspecific manner because the luminescence of the upconversion fluorescent nanoparticle was effectively strengthened in the presence of bacteria. When the developed nanosensor was applied to quantify multiple bacteria including Escherichia coli, Salmonella, Cronobacter sakazakii, Shigella flexneri, Vibrio parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes, a linear range of 103 to 108 cfu mL-1 and a detection limit of 1.30 × 102 cfu mL-1 have been obtained for the seven model mixture bacteria. In addition, the similar linear range and detection limit were also obtained for the detection of single bacteria. The present approach also exhibited acceptable recovery values ranging from 70.0 to 118.2% for bacteria in real samples (water, milk, and beef). All these results suggested that the guanidine-functionalized upconversion fluorescent nanosensor could be considered as a promising candidate for the rapid detection and surveillance of microbial pollutants in food and water.