Abnormal colonic features, such as excessive generated reactive oxygen species (ROS) and increased epithelial oxygenation, result in the overproliferation of harmful aerobic bacteria, which promotes intestinal dysbiosis and exacerbates the progression of colitis. Conventional therapies include the use of drugs to control the inflammatory response or biologic agents (e.g., probiotics) to modulate intestinal microflora. However, current therapies usually suffer from severe side effects, poor bioavailability, and insufficient efficacy. Inspired by the oxygen depletion of spore germination in response to nutrient germinants, we constructed a microfluidic-derived pathological microenvironment-activated spore nanozyme germinator for the precise regulation and treatment of colitis. The prepared spore nanozyme germinator significantly downregulates ROS to alleviate intestinal inflammation and efficiently promotes the germination of spores at the sites of colitis to rapidly deplete colonic oxygen, thus inhibiting harmful Enterobacteriaceae bacteria and restoring intestinal microflora homeostasis. With this, the multifacet values of the spore-based device to precisely regulate colitis are demonstrated. This work provides a robust platform for developing advanced and precise living spore-based biologics across broad biomedical applications.