In legume plants, uricase gene (Nodulin-35) plays a positive role in metabolism of ureide and amide compounds in symbiotic nitrogen-fixing in the nodules. In this study, a pot experiment was performed to examine the effects of ammonium application on the transcription of MsU2 gene and distribution of major nitrogen compounds in alfalfa Medicago sativa. Data showed that alfalfa plant has a significant difference in contents of nitrogen compounds in xylem saps compared with soybean plant, and belongs to typical amide type legume plants with little ureide accumulation, and the accumulation of asparagines and ureide in the tissues of alfalfa is mainly gathered in the nodules. Northern blotting showed that excessive ammonium significantly inhibited the transcription of MsU2 gene in the nodules and roots, and mRNA accumulation of MsU2 gene in the plants exposed to excessive ammonium decreased gradually with culture time extension, indicating that application of ammonium significantly inhibited the transcription of MsU2 gene in the alfalfa plants. Although the application of excessive ammonium increased the contents of amino acids in various tissues of alfalfa, the accumulation of allantoin reflecting the strength of uricase activity is remarkably reduced in the xylem saps, stems and nodules when alfalfa plants exposed to excessive ammonium, suggesting that application of excessive ammonium generated a negative effect on symbiosis fixing-nitrogen system due to inhibition of ammonium ion on uricase activity in the nodules of alfalfa. This result seems to imply that application of excessive ammonium in legume plants should not be proposed to avoid affecting the ability of fixing nitrogen in the nodules of legume plants, and reasonable dose of ammonium should be recommended to effectively utilize the fixed N from atmosphere in legume plant production.