The invention discloses a determination method of related substances of mesalazine enteric-coated sustained release tablets. The method includes: performing gradient elution by adopting a C18 chromatographic column and preparing a mobile phase with a dipotassium phosphate salt solution, a TBAH solution and a methanol solution, wherein the dipotassium phosphate salt solution adjusts PH to 7.2 through phosphoric acid, and the TBAH solution adjusts PH to 7.5 through a sulfuric acid solution (1[rightward arrow]2). According to the method, impurities of mesalazine are fully revealed, related substances of mesalazine can be rapidly detected, the security of the product can be improved, the method is scientific and reliable, and related substances of mesalazine can be controlled.