Nalbuphine was a semisynthetic opioid analgesic widely used in the treatment of both acute and chronic pain. We developed and validated a rapid, simple and sensitive method by ultra-performance liquid chromatography-tandem mass spectrometry (MS/MS) for the simultaneous quantitation of nalbuphine in human plasma, and we reported the pharmacokinetic features of patients during general anesthesia for abdominal surgery. Sample separation was achieved on a Kinetex Phenyl-Hexyl column (50 × 2.1 mm, 1.7 μm) after simple protein precipitation with acetonitrile. The mobile phase was composed of acetonitrile and 3 mM of ammonium acetate aqueous solution with 0.1% formic acid. Gradient elution was used in 4.5 min with a flow rate of 0.5 mL/min at 40°C. MS detection using AB Sciex QTRAP 5500 mass spectrometer was characterized by electrospray ionization for positive ions in multiple reaction monitoring mode. Quantitative ion pairs were m/z 358.4 → 340.1 for nalbuphine and m/z 340.0 → 268.3 for nalmefene, which were used as the internal standard (IS). The calibration curves showed good linearity (r2>0.99) over concentration range of 0.1-500 ng/mL. The intra-and inter-batch precisions were within 10.67%, and accuracy ranged from 94.07 to 105.34%. The IS-normalized matrix factors were 1.02-1.03 with RSD% (≤5.82%). The recoveries ranged from 101.09 to 106.30%. In conclusion, a rapid, simple, sensitive and economical analytical method was developed and validated to detect the concentration in plasma samples obtained from patients receiving nalbuphine intravenous injection and was successfully applicated to human pharmacokinetic studies of nalbuphine.