A novel method for the extraction of inosinic acid in Smoked Katsuwonus pelamis based on water was developed. The inosinic acid was extracted by 95 degrees C water for 5 min. The subsequent analysis of inosinic acid was achieved on an Agilent Zorbax SB-C18 column (150 mm x 4.6 mm, 5 microm) with the mobile phase of methanol-50 mmol/L ammonium acetate (5: 95, v/v) at a flow rate of 1.0 mL/min at 30 degrees C. The detection wavelength was set at 254 nm. The results showed that the linear range for inosinic acid in Smoked Katsuwonus pelamis was between 1.0 mg/L and 120.0 mg/L with the limit of detection (LOD, S/N = 3) of 4 mg/kg and the limit of quantification (LOQ, S/N = 10) of 12 mg/kg. At the spiked levels of 0.40, 2.00, 4.00 g/kg (n = 3), the recoveries ranged from 84.6% to 100.2% with the relative standard deviations (RSDs) between 1.00% and 6.12%. Compared with traditional extraction method by perchloric acid, the extraction efficiency was improved by 37.0%. The novel method is safe, stable and accurate for the determination of inosinic acid in Smoked Katsuwonus pelamis, which can also be applied to the determination of inosinic acid in other foods and deserved to be spread.