In enzymology, a dimethylglycine oxidase (EC 1.5.3.10) is an enzyme that catalyzes the chemical reaction

N,N-dimethylglycine + H₂O + O₂ ${\displaystyle \rightleftharpoons }$ sarcosine + formaldehyde + H₂O₂

The 3 substrates of this enzyme are N,N-dimethylglycine, H₂O, and O₂, whereas its 3 products are sarcosine, formaldehyde, and H₂O₂.

This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-NH group of donors with oxygen as acceptor. The systematic name of this enzyme class is N,N-dimethylglycine:oxygen oxidoreductase (demethylating). It employs one cofactor, FAD.

As of late 2007, 3 structures have been solved for this class of enzymes, with PDB accession codes 1PJ5​, 1PJ6​, and 1PJ7​.