Urine, an easily accessible body fluid, contains rich metabolic compounds reflecting the human health condition. Though urine analysis can be carried out by many conventional analytical methods, direct, rapid, and simultaneous identification of a variety of urinary metabolites by a handheld device remains challenging. Recent developments in nanopore small molecule sensing may suggest direct urine analysis performed by an engineered nanopore. Here, a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore immobilized with a single nickel ion is confirmed to be suitable for the simultaneous identification of six urinary metabolites: creatinine, taurine, histidine, 1-methylhistidine, 3-methylhistidine, and hypoxanthine. With the assistance of machine learning, an overall accuracy of 98.5% was reported. This urine sensing capacity was further applied in real-time urinary metabolite monitoring after diet and physical exercise. These proofs of concept suggest a variety of nanopore-based body fluid analyses for early diagnosis, health management, pharmacokinetic monitoring, and doping tests.