Pathogenic infections may lead to disruption of homeostasis, thus becoming a serious threat to the human health. Understanding the interactions between bacteria and macrophages is critical for therapeutic development against sepsis or inflammatory bowel disease. Here, we report a technique using droplet biosensors for the detection of nitric oxide (NO) secreted by a single macrophage under inflammatory stimuli. We demonstrated that the limit of detection can be promoted more than two orders of magnitude by our approach, in comparison to the conventional microplate format. The experiments of co-encapsulating single macrophages and different numbers of Escherichia coli (E. coli) enabled fluorescence monitoring of NO secretion by single macrophages over the incubation, and investigation of their interactions inside the isolated droplet for their separate fates. Our approach provides a unique platform to study the bacteria-macrophage interactions at the single cell level.