This study presents a simple urease method for analysis of ammonia and urea in freshwater aquaculture systems. Urea is hydrolysed into ammonia using urease followed by analysis of released ammonia using the salicylate-hypochlorite method. The hydrolysis of urea is performed at room temperature and without addition of a buffer. A number of tests were performed on water samples obtained from a commercial rainbow trout farm to determine the optimal urease concentration and time for complete hydrolysis. One mL of water sample was spiked with 1.3 mL urea at three different concentrations: 50 μg L−1, 100 μg L−1 and 200 μg L−1 urea-N. In addition, five concentrations of urease were tested, ranging from 0.1 U mL−1 to 4 U mL−1. Samples were hydrolysed for various time periods ranging from 5 to 120 min. A urease concentration of 0.4 UmL−1 and a hydrolysis period of 120 min gave the best results, with 99.6–101% recovery of urea-N in samples spiked with 100 or 200 μg L−1 urea-N. The level of accurate quantification of ammonia using the method is 50 μg L−1 NH4+-N, and the detection level is 5–10 μg L−1 NH4+-N.