There is a global concern regarding the increasing pollution of natural water bodies by nitrate as a result of anthropogenic activities. To address this situation, natural organic solid substrates (NOSS) have been investigated as carbon sources in denitrification. Despite advances in this field, a lack of knowledge about the kinetics involved in the denitrification processes using NOSS still exists. For this reason, we have studied denitrification using Saccharum spontaneum L. (S. spontaneum) as NOSS to better understand these kinetic processes. This paper presents experimental results obtained for the release of organic carbon from S. spontaneum, and for its use by denitrifying bacteria. Laboratory tests were developed under anoxic conditions in batch reactors. Our results showed that the kinetics of release of organic carbon from the S. spontaneum was first order (0.08 d−1). Furthermore, the maximum rate of utilization of substrate (5.61 mg N-NO3− mgVSS−1 day−1) and the denitrification rate (327 mg N-NO3−L−1 d−1) were high (VSS = volatile suspended solids). We demonstrated that it is possible to obtain high yields of denitrification using organic carbon released from S. spontaneum. This study improves the knowledge on the use of NOSS, as alternative sources of carbon for denitrification.