The ability to suppress soil nitrification through the release of nitrification inhibitors from sorghum roots is termed “biological nitrification inhibitor” (BNI). It was reported that the release of BNI from sorghum roots under ammonium nutrition was much more than under nitrate nutrition. It is not understood whether the nitrogen metabolism or the rhizosphere pH in relation to the uptake of ammonium and nitrate is the effect on the release of BNI. In this study, sorghum plants were cultivated in sands with ammonium or nitrate. The root medium pH was controlled by pH-stat system and root exudation was collected to test the BNI activity and release rate by gene modified Nitrosomonas with florescence. The results showed that nitrogen form was the major factor which affected the release of BNI. Moreover, under the pH 6.0, the release of BNI was related to the release of proton by sorghum roots. Our study indicated the release of BNI by sorghum roots under ammonium nutrition rather than under nitrate nutrition might be a potential to suppress ammonium oxidation in soil, which could improve nitrogen use efficiency (NUE).