Abstract: A method was established for the simultaneous determination of 9 macrolide antibiotics in seawater by liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry. Empore C₁₈ solid phase extraction film was used to extract macrolide antibiotics from seawater. The analytes were seperated by Hypersil GOLD C₁₈ chromatographic column and gradient eluted with the mixtures of acetonitrile and 0.005 mol·L^-1 ammonium acetate solutioncontaining 0.1% (φ) formic acid, and analyzed in full scan/data dependent MS² (Full MS/dd-MS²) mode. The results showed that the retention times of 9 macrolide antibiotics were all within 13 min. Linearity relationships between values of mass concentration of 9 macrolide antibiotics and their peak area responses were kept in the range of 1-500 μg·L^-1, with detection limits (3S/N) in the range of 0.02-0.12 ng·L^-1. Recovery was tested by standard addition method using a negative seawater sample as matrix at the 3 concentration levels, giving values of recovery in the range from 79.0% to 105% and of RSDs (n=6) of determined values lower than 7.5%. The proposed method was applied to the determination of 9 macrolide antibiotics in 25 samples of seawater, and ROM, CLM, AZM, and TMC were detected in varying degrees.