OBJECTIVE To develop a method to determine the drug release of Esomeprazole Magnesium Enteric-coated Tablets in vitro, and investigate its drug release behavior. METHODS Dissolution of Esomeprazole Magnesium in Esomeprazole Magnesium Enteric-coated Tablets were determined by RP-HPLC with Isocratic method. In the environment of simulating people′s gastrointestinal tract dissolution medium, The drug release consistency in vitro between Homemade preparations and Original development agents was compared. RESULTS A good linearity was shown when the contents of Esomeprazole Magnesium were at 5.025-50.25 μg · mL-1, and r value was 0.9999, resp. The average recoveries were at 99.1%-99.6%. RSDs were less than 2.0%, resp. (n = 3). LOQ was 0.06 μg · mL-1. The release of Homemade preparations in vitro was similar to Original development agents. CONCLUSION Esomeprazole Magnesium Enteric-coated Tablets and Original development agents display a similar drug release behavior in vitro, which can provided a guarantee for the late bioequivalence trials research.