The variation of the integrity of the intestinal barrier is closely related to the occurrence and progression of various diseases, making its accurate detection and monitoring essential to provide reliable information for guiding the refinement of treatment scenarios. Herein, we developed a strategy utilizing glutathione-capped gold clusters (Au-GSH) to achieve simultaneous in vivo second near-infrared (NIR-II) fluorescence imaging and in vitro colorimetric urinalysis for noninvasive detection and monitoring of intestinal barrier integrity. After oral administration, Au-GSH can demonstrate different distribution behaviors in terms of the variation of intestinal barrier integrity. Specifically, Au-GSH could selectively permeate through compromised intestinal barrier to be distributed into the bladder in view of its ultrasmall size below the glomerular filtration cutoff, leading to the rapid excretion through urine. Such process can be visually profiled by collecting NIR-II fluorescence (>1100 nm) emitting from Au-GSH in a noninvasive real-time manner. By virtue of the peroxidase-like activity of Au-GSH, the simple colorimetric urinalysis was further established for evaluating the integrity of the intestinal barrier, advancing the detection and monitoring performance. As a result, our developed strategy successfully detected the damage of intestinal barrier integrity in ulcerative colitis (UC) mice, a common disease characterized by compromised intestinal barrier integrity. Excitingly, the monitoring of restoration of intestinal barrier integrity in both therapeutic and preventative modes for UC mice was also realized by our strategy, making it a promising diagnosis scenario for diseases related to the variation of intestinal barrier integrity.