We present here an easy-to-operate and efficient method for enzyme and inhibition assays of urease, which is a widely distributed and important enzyme that catalyzes the hydrolysis of urea to ammonia and CO2. The assay was achieved by integrating CE technique and rapid on-line derivatization method, allowing us to continuously drive the sample to the capillary, thus to measure the amount of the product ammonia from the beginning to the end of the reaction. The method exhibits excellent repeatability with RSD as low as 2.5% for the initial reaction rate (n = 5), with the LOD of ammonia of 20 mu M (S/N = 5). The enzyme activity as well as the inhibition of urease by Cu2+ were investigated using the present method. The results show that Cu2+ is a noncompetitive inhibitor on urease, in accordance with the result published in the literature. The enzyme activity and inhibition kinetic constants were obtained and were found to be consistent with the results of traditional off-line enzyme assays. Our study indicates that the present approach is a reliable and convenient method for analysis of the urease activity and inhibition kinetics by continuous on-line monitoring of the ammonium formation based on CE.