A method for the determination of characteristic compound 3,5-dime thoxybenzoate- 4-diglucoside (leptosperin) in Manuka honey was developed by automatic on-line solid phase extraction-liquid chromatography-high resolution mass spectrometry (SPE-LC-HRMS). The samples were separated on a Dikma Diamonsil Plus C18 column (150 mmx4. 6 mm, 5 |xm) using the mobile phases of 0. 1% (v/v) formic acid aqueous solution and acetonitrile with gradient elution. The compound was detected with negative electrospray ionization (ESI-) in Target-MS2 mode. The results showed that the linear range was 0. 5-100. 0 mg/L, the correlation coefficient was 0. 999 3. The limit of detection (LOD, S/N 5= 3) and limit of quantification (LOQ, S/N5= 10) of the method was 3 mg/kg and 10 mg/kg, respectively. The recoveries at the spiked levels of 50. 0, 100. 0, 200. 0 mg/kg (10. 0, 20. 0, 50. 0 mg/kg in black locust samples) were in the range of 82. 0%-95. 2% with the relative standard deviations ranging from 2. 7% to 9. 7% (n = 6). The proposed method was applied to 95 mature honey samples from hives in New Zealand including 12 different kinds and 50 commercial honey samples from four different countries. The method is fast, sensitive and accurate to provide technical support to solve the judgment of the Manuka honey imported from New Zealand.